WO1997038005A1

WO1997038005A1 - Novel antibiotic, process for the preparation of the same, and drug composition containing the same

Info

Publication number: WO1997038005A1
Application number: PCT/JP1997/001221
Authority: WO
Inventors: Kenzo Harimaya; Yoshikazu Sato; Fukio Konno; Toru Sasaki; Mina Kanda; Noriko Chiba; Takashi Mikawa
Original assignee: Meiji Seika Kaisha, Ltd.; Mitsubishi Chemical Corporation
Priority date: 1996-04-10
Filing date: 1997-04-09
Publication date: 1997-10-16

Abstract

An antifungal and antilipemic agent comprising substance MK6059 of the following general formula (I) which is prepared by culturing a MK6059 strain belonging to the genus Ellisiodothis in a conventional nutrient medium for microbes, and subjecting the resulting system to extraction with solvent, ion exchange, silica gel chromatography, gel filtration chromatography or the like to isolate the objective substance.

Description

TECHNICAL FIELD The present invention relates to a novel antibiotic MK 6059 having an antifungal / hyperlipidemic activity and its antifungal agent and hyperlipidemia. The present invention relates to a use as a remedy for diseases and a method for producing the same. BACKGROUND ART Conventionally, various physiologically active substances produced by microorganisms are known and have been put to practical use in the fields of pharmaceuticals, cosmetics, veterinary drugs, agricultural chemicals, etc., but have more useful activities than these known compounds. The emergence of new substances is always desired.

The antibiotic MK 6059 substance according to the present invention is considered to be a triterpene or steroid, and as compounds similar thereto, PF 1032 (Japanese Unexamined Patent Publication (Kokai) No. 3-119993), eryloside ACC amerly et al., J. Nat Prod. 52, No. 1. 167-170, 1989) are known, and MK 6059 substances are clearly distinguished from these known substances due to their different physicochemical properties. DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel substance having a more useful activity than various known physiologically active substances produced by microorganisms.

The present inventors have found that a substance having an antifungal and antihyperlipidemic activity is produced in a culture of one strain belonging to the genus Ellis iodothys. The present invention has been completed by isolating the active substance produced by this strain and clarifying its physicochemical properties.

The gist of the first invention is a substance represented by the following formula (I) and a salt thereof. The substance represented by formula (I) is a new substance and is named MK 6059 substance.

The gist of the second aspect of the present invention is to culture a microorganism belonging to Aspergillus oryzae and capable of producing the MK609 substance, and collecting the MK609 substance from the culture. It is in the method of producing the characteristic MK 6509 substance.

The gist of the third aspect of the present invention is a pharmaceutical composition containing an MK 6059 substance as an active ingredient. Uses of the pharmaceutical composition include antifungal agents and therapeutic agents for hyperlipidemia. Hereinafter, the present invention will be described in detail.

<1> MK 659 substance of the present invention

The physicochemical and biological properties of the MK 659 substance according to the present invention are as follows c

1. Physical and chemical properties of MK 6 0 5 9 substances

(1) Elemental analysis value:

Carbon 6 5.8 9%

Hydrogen 8.78%

(2) Molecular weight:

64 6 (FD -MS, m / z 64 7, H ⁺ )

(3) Molecular formula:

C 37 H 58 ^ 9

(4) Specific rotation:

[al D = + 5 8. 7 7 (c = 1. 0, menoru) (5) Ultraviolet absorption spectrum:

End absorption

(6) Infrared absorption spectrum:

The spectrum measured with a bromide-resistant pill is as shown in FIG.

(7) Hydrogen nuclear magnetic resonance spectrum:

The 400 MHz hydrogen nuclear magnetic resonance spectrum measured in the heavy methanol solution is as shown in FIG.

(8) Carbon nuclear magnetic resonance spectrum:

The 100 MHz carbon nuclear magnetic resonance spectrum measured in a heavy methanol solution is as shown in FIG.

(9) Solubility:

Soluble in black form, getyl ether, acetone, ethyl acetate, methanol and ethanol. Insoluble in n-hexane and water.

(10) Basic, acidic, neutral:

Weakly acidic substance The planar structure of the MK650 substance represented by the formula (I) discovered by the present inventors is represented by the mass spectrum, UV spectrum, IR spectrum (FIG. 1), Confirmed from the NMR spectrum (Figs. 2 and 3).

<2> Production method of MK 6 0 5 9 substance

The MK 609 substance with the above structure belongs to the Aspergillus oryzae and is used to culture microorganisms capable of producing the MK 609 substance, and the MK 609 substance is collected from the culture. It is obtained by doing. Microorganisms belonging to the genus Erythodorosis are examples of the microorganisms belonging to the micrococcus. Further, as a microorganism belonging to Erythododosis II, E11 isi0 dothisinquinans is mentioned, and as a specific strain, E. coli incluence L12 isolated by the present inventors is used. There are 27 strains. The bacteriological properties of the L1227 strain are shown below. 1. Mycological properties of MK 6059 substance producing bacteria

Pseudopods are scattered or clustered on the host plant and are superficial, having a semicystic shell shape, diameter of 300-600; um, height of 50-80〃m, and A small circular hole opens.

The upper lid and the rim consist of cells arranged radially.

The shell wall is 15 to 20 m thick and consists of 3 to 4 layers of polygonal or square cells. The outer side is black-brown, the 內 side is light brown, and a thin film.

A large number of children have a false side thread, they have an inverted-cylindrical to cylindrical inverted-rod shape, and have a size of 58.5 to 66.0 x 26.4 to 33.0 ^ m and a short base. It has a stalk, is thick-walled, has a double-walled structure, and is eight-spore.

The false side thread is thread-like, has partition walls, and is colorless.

Ascospores are arranged in one row above the asci and two rows below. The shape is elliptical, the size is 15.6 to 18.4 9.3 to 10. O ^ m, one cell, colorless and smooth.

2. Characteristics of culture on various media

B) Potatoes cultured on glucose agar medium (PDA) for 27, 10 days The colonies spread to 5-7 cm in diameter in 10 days. The color initially changes from white to off-white, and gradually to brown-grey. The back of the colony is yellow-brown. The basal hypha elongates radially, is branched, reaches 6 m in width, and is colorless. Aerial hyphae are abundantly formed. No anamorphic and teleomorphic reproductive organs were observed on the PDA.

Mouth) Culture on malt agar medium (MA) for 27, 10 days

The characteristics of the culture on this medium almost coincided with the properties on the PDA.

3. Physiological properties

B) Optimal growth conditions

Optimum pH: 6-7 (can be transplanted to production medium after cultivation in LCA liquid medium for 14 days)

Optimal temperature: 25 to 27 (can be transplanted to production medium after 10 days of culture on PDA agar) Mouth) Allowable growth conditions

pH: 4-9 (can be transplanted to production medium after cultivation in LC A liquid medium for 14 days)

Temperature: 20-30 (can be transplanted to production medium after 10 days of culture on PDA agar)

4. Taxonomic considerations

B) higher taxonomic position

This strain (L1227 strain) grows on the surface of dicotyledonous plants to form an upper lid-like pseudo-joss shell, has a small circular hole at the center of the upper lid, the upper lid and its periphery Form asci between persistent pseudocysts, composed of cells arranged radially. The ascus is thick and double-walled. Based on these characteristics, we searched according to the classification system of ES Luttre 11, Locouloasco mycetes, The Fungi, vol. 4 A (ed.GC A in swo rthetal. As a result, the bacterium (L1227) was found to be a member of the class Microcylia (Microcrothyriaceae) of the order Mycosphacomyceae (Loculoasco mycetes) and the order Hemis isphaeriales. Mouth) Identification at the genus and species level

According to the classification of Microt hy riaceae in E.S.Luttrell, Locouloasco mycetes, i, he Fun gi, vol.4 A (ed.GC A in swo rthetal.) 203-205 (1973) This family includes 23 genera. Of the 23 genera, there are My iocopron genus and E11 isiodothis genus while the spores are single chamber. These two genera are distinguished by the manner in which pseudo-shell-forming hyphae enter host cells. In other words, the cells that infect host cells by wedge-shaped hypha are distinguished from the genus Myi0c0pr0n, and the genus in which normal hyphae invade host cells is distinguished from E1 isiodothis. When a cross section of this strain was prepared and the state of hyphal invasion into the host cells was observed, the Myococ 0 pron-type wedge-like hyphal morphology was not observed. Therefore, the book The strain was determined to be a strain belonging to the genus E is] isiodothis. According to Die G attungender ame rosporen Pyren omy ceten, B eitr, K ryptog ame nf 1. S chweiz 11 (1); 95-98 of von A rx and E. Miiller (1 954), Seven species of l 1 isiodothis are known (E. inquinans, E. sm i) acis, E. microdisca, E. pittieri, E. gra mm atophy 1 1 i, E. pandani, E. re hm iana). Each type is classified mainly by the shape and size of the asci and ascospores. The properties of this bacterium were compared with these various properties.The bacterium of this bacterium was 58.5-66.0 x 26.4-33.0 m, and the ascospores were 15.6-18.4. x 9.3-: 10.0 m in size, almost equivalent to E. inquinans (children 40-55 x 18-25 m, spores 14-: L 7 x 6-8 m) Matched. Therefore, this strain was identified as a strain belonging to E11 isiodothisinquinans (Elicidochis cis, incuinuinance), and named as Elicidociscis incinuinance L1227. FERM was registered on February 27, 1996 by the Ministry of International Trade and Industry, Ministry of International Trade and Industry, National Institute of Bioscience and Human Technology (Postal Code 305, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan). Deposited under the accession number P-15479, transferred to an international deposit under the Budapest Treaty on March 28, 1997, and deposited under the accession number FERM BP-5889.

The L1227 strain is susceptible to change in properties as seen in other molds. For example, any L1227 strain or a mutant strain (naturally occurring or inducible) derived from this strain, a transzygote or a genetically modified product that produces an MK6059 substance can be used in the present invention. .

Culture method of CMK6059 substance-producing bacteria)

In the present invention, the above-mentioned bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As a nutrient source, glucose, water syrup, dextrin, sucrose, starch, sugar, animal and vegetable oils and the like can be used. As a nitrogen source, soybean flour, wheat germ, corn steep 'liquor, cottonseed kashiwa, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary, it is effective to add sodium, potassium, calcium, magnesium, konokoroto, chlorine, phosphoric acid, sulfuric acid and other inorganic salts capable of generating ions. Organic and inorganic substances can be added as appropriate to support the growth of bacteria and promote the production of MK 6059 substance.

As a culture method, a culture method under aerobic conditions, particularly a submerged culture method is most suitable. Suitable temperature for cultivation is 10 to 30 ° C. In most cases, culturing at 20 to 27 _c Production of MK6059 substance varies depending on the culture medium and culture conditions, but usually 3 to 10 for shaking culture and tank culture. Its accumulation reaches the highest during the day.

When the amount of accumulation in the culture reaches the maximum, stop the culture and isolate and purify the target substance from the culture solution.

Purification method of CMK6059 substance)

Since the MK6059 substance obtained by the present invention is a fat-soluble substance, isolation and purification of the MK6059 substance from the culture can be performed by utilizing its properties. Synthetic adsorbents such as Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation); gel filtration agents such as Sephadex LH-20 (manufactured by Pharmacia); Toyopearl HW-40 (manufactured by Tohso Corporation); ethyl acetate; A solvent extraction method using a mouth form or the like, column chromatography using silica gel, alumina, or the like, and a preparative thin-layer mouth chromatography using silica gel as a carrier are effective.

A high-purity MK6059 substance having the above-mentioned physicochemical properties can be obtained by the above methods or by appropriately combining them.

In testing various MK 6059 substances in various purification processes, Candidapseudotropicalis M9035 was used as a test bacterium, and a paper disc was prepared according to the method submitted by the Society of Chemotherapy. Perform by law. On agar medium by paper disg method The growth-inhibition circle of 10 shows a linear relationship with the log of the concentration of 10-250 / g / ml, giving an inhibition circle of 15-25 mm.

<3> Pharmaceutical composition of the present invention

The pharmaceutical composition of the present invention contains an MK 659 substance as an active ingredient. Uses of the pharmaceutical composition include antifungal agents and therapeutic agents for hyperlipidemia.

When the MK609 substance is used as a drug, it can be made into a formulation suitable for the administration route together with a usual formulation carrier. For example, for oral administration, it is prepared in the form of tablets, capsules, granules, powders, liquids and the like. In preparing a solid preparation for oral administration, conventional excipients, binders, lubricants, other coloring agents, disintegrating agents and the like can be used.

Excipients include, for example, lactose, starch, talc, magnesium stearate, crystalline cellulose, methylcellulose, carboxymethylcellulose, glycerin, sodium alginate, gum arabic, etc., and binders such as polyvinyl alcohol, polyvinyl ether , Ethylcellulose, gum arabic, shellac, sucrose and the like, and examples of the lubricant include magnesium stearate, talc and the like.

In addition, conventionally known coloring agents and disintegrating agents can also be used. The tablets may be coated by a known method. The liquid preparation may be an aqueous or oily suspension, solution, syrup, elixir or the like, and is prepared by a commonly used method. When preparing an injection, a PH regulator, a buffering agent, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the compound of the present invention, and a subcutaneous, intramuscular, or intravenous injection is prepared by a conventional method. Can be manufactured. As a base for producing a suppository, for example, an oleaginous base such as cocoa butter, polyethylene glycol, lanolin, fatty acid triglyceride, and witebsol (registered trademark Dynamite Nobel) can be used.

The dosage of the preparation thus prepared varies depending on the patient's symptoms, weight, age, etc., and cannot be taken uniformly.However, the compound of the present invention is usually about 1 to 200 mg / day per adult per day. The amount should be in the range of 1 to 4 times a day. It is preferably provided. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing an IR spectrum of MK6059 substance by the KBr method. FIG. 2 is a diagram showing a 40 nm NMR spectrum of MK6059 substance in deuterated methanol.

FIG. 3 is a view showing a 100 MHz ¹³ C NMR spectrum of MK 6059 substance in deuterated methanol. BEST MODE FOR CARRYING OUT THE INVENTION Examples of the present invention will be described below.Since the properties of the MK 6059 substance have been clarified by the present invention, various methods for producing the MK 6059 substance are described based on those properties. Can be devised. Therefore, the present invention is not limited to the examples, and the MK 6059 substance is produced by applying known means based on the properties of the MK 6059 substance revealed by the present invention as well as the modification means of the examples. It covers all methods for concentration, extraction and purification. Example 1 Culture of Erythodorosis incubinus L1227 strain Glucose 5.0%, soybean flour 1.0%, meat extract 0.4%, peptone 0.4%, yeast powder 0.1%, NaCl 0.1% A seed medium (pH 7.0) containing 25% and CaCO ₃ 0.5% was dispensed into 10 20 Om 1 Erlenmeyer flasks each with 4 Om 1 and autoclaved for 121 and 20 minutes.

Next, one loopful of the L1227 strain was inoculated with a platinum loop, and cultured with shaking at 210 rpm at 26 for 4 days.

Separately, water syrup 2.0%, soy flour 1.0%, soybean oil 0.15%, sun grain 0.25%, cottonseed meal 0.5%, iron sulfate7 hydrate 0.0005%, Prepare a main fermentation medium (ρΗ6.0) containing 0.00005% nickel chloride hexahydrate, 0.0005% cobalt chloride hexahydrate and 0.196 calcium carbonate, and add 8 Om I of each. Dispense into 100 500m〗 Erlenmeyer flasks and autoclaving at 121 for 20 minutes did. This main fermentation medium was inoculated with 4 ml of the seed culture solution, and cultured with shaking at 210 rpm for 4 days at 26 days. The obtained culture was centrifuged to obtain a culture supernatant and cultured cells. Example 2 Purification of MK 6059 substance 6.0 L of the culture supernatant obtained in Example 1 and the cells were extracted with 6.6 L of 70% acetone water for 1 hour at room temperature, The removed cell extract was concentrated and the cell extract concentrate obtained by concentrating 1.5 L was combined to make 7.5 L, and extracted with the same amount of ethyl acetate. The extract was washed with water, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 4.47 g of an oily substance.

Spray 4.5 g of diatomaceous earth on the resulting oily substance, dry overnight under reduced pressure, and place on a 200 ml column of silica gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) filled with chloroform. After washing with form, then form-form methanol mixture (100: 1) and form-form-methanol mixture (50: 1), form form-methanol mixture (20: 1), form-form-methanol mixture (20: 1) Chromatography developed at 10: 1) was performed.

The active fraction was collected and concentrated to dryness under reduced pressure to obtain 304 mg of an oily substance. This oily substance was dissolved in a small amount of methanol, placed on a 800 ml column of Sephadex LH-20 (Pharmacia) filled with methanol, and developed with methanol.

The active substance was eluted in fractions 30 to 34 in 14 ml fractions. This active fraction was collected and concentrated to dryness under reduced pressure to obtain 246 mg of an oily substance.

This oily substance is dissolved in a small amount of methanol and filled with methanol. (Manufactured by TOSOH CORPORATION) Placed on a 1 L tower and developed with methanol. The active substance eluted in fractions No. 45-48 in 10 ml fractions. The active fractions were collected and concentrated to dryness under reduced pressure to obtain crude MK 6059 substance. Dissolve 162 mg of this crude MK 6059 substance in a small amount of methanol and place it in a 20 ml column of Cosmoseal 75 C18-0PN (manufactured by Nacalai Tesque) filled with 60% methanol, and add 60% methanol 250m 1-100% methanol 250ml A gradient elution was performed. The active substance eluted in fractions No. 37-42 in the 5 g fraction.

The oily fractions were collected, concentrated to dryness under reduced pressure, and 89 mg of purified MK605 9 substance was obtained as a colorless powder. The physicochemical properties of this substance are as described above.

The active fraction in each purification step was measured by the paper disc method using Candida * Pseudotropicalis M 9035 as a test bacterium. Test Example 1 Antifungal activity

Table 1 shows the antifungal spectrum of the MK6059 substance against various fungi. The methanol solution of each concentration of the MK6059 substance was infiltrated into a paper disk in an amount of 20 u1 at a time, and indicated by the diameter of the inhibition circle measured by the agar plate method. The test bacteria in Table 1 are as shown below.

1. Saccharomyces, Selepiche S HY 3

(Saccharomyces cerevisiae SHY3)

2. Candida << Albicans M9001

(Candida albicans M9001)

3. Candida pseudotropicalis M9035

(Candida pseudotropical is M9035) Table 1 Antifungal spectrum of MK 6059 Concentration Blocking circle diameter (mm)

(fi g / m 1) Bacteria 1 Bacteria 2 Bacteria 3

1000 24.6 27.8 25.8

250 21.7 25.5 25.0

62.5 18.9 22.8 20.6

12.6 15.3 16.4 16.5 As described above, the MK 6059 substance has antifungal activity and is expected to be applied as an antifungal agent. Pharmacological Test Example 1 Cholesterol-lowering effect (1) A test drug suspended in 0.5% CMC was added to mice (6 mice / group) weighing around 19 g preliminarily bred for one week, and 0.1 g of 10 g of the test drug suspended in 0.5% CMC. Oral administration to body weight once daily in the evening for 5 days. After the administration on the 5th day, the mice were fasted overnight, the test drug was orally administered the next morning, and 3 hours later, blood was collected by decapitation and blood serum was separated. Serum total cholesterol (Tch) was determined using a kit of cholesterol C-Test Co., Ltd. (Wako Pure Chemical Industries), and triglyceride (TG) using a kit of triglyceride G-Test Co., Ltd. (Wako Pure Chemical Industries). . The drug dose was 10 Omg / kg for the MK 6059 substance, 30 Omg / kg for the clofibrate, and 0.5% CMC was administered to the control group. Table 2 shows the results. Table 2 Effect of MK 6059 on serum lipids in normal mice Weight gain Serum lipids (mg¾i)

Treatment (g) Tch TG control 4.5 ± 0.5 137.4 Sat 6.6 89.6 ± 7.8 K6059 100 mg / kg.po 4.7 ± 0.5 102.4 Sat 3.4 * 95.0 ± 7.8 D Dif 'rate 300 mg / kg.po 4.2 ± 0.6 118.8 ± 13.3 92.4 ± 9.9

* N = 6, P <0.01

The MK6059 substance significantly reduced the total cholesterol level in the serum of normal mice as compared with the control group by continuous oral administration of 10 OmgZkg for 5 days. No significant improvement was observed with clofibrate 300 mg / kg for 5 consecutive days. Pharmacological Test Example 2 Triglyceride lowering effect—effect on liver weight The test drug was suspended in 0.5% CMC and orally administered once daily at 10 Omg / kg.

After completion of oral administration 4 days later, the mice were fasted, the test drug was orally administered the next day again, blood was collected 4 hours later, serum was separated, and serum lipids were quantified. The liver was taken out immediately after blood collection, and its weight was measured. The results are shown in Table 3 as values per 10 g of mouse body weight. Table 3 Effect of continuous administration (5 days) of MK 6059 substance on serum lipids of normal mice Serum lipid (mgl¾) Liver weight treatment Tch TG g / 10g bw Control 131.6 ± 10.6 159.6 Sat 4.5 0.45 Sat 0.01 605ΜΚ117.2 Sat 6.1 120.1 ± 11.Γ 0.43 Sat 0.02 lOOrag / kg.po, 5days

Ku nfif'let 140.1 Sat 6.3 102.0 Sat 3.6 '0.54 Sat 0.01

400mg / kg. P.o., 5days

Ν = 5, * Ρ <0. 05, * * Ρ <0. 01

The # 6059 substance significantly reduced triglycerides in serum in normal mice at a dose of 10 Omg / kg for 5 consecutive days. At this time, no effect on liver weight was observed.

The control drug, clofibrate, at 8 to 400 mg / kg for 5 consecutive days significantly reduced triglyceride, but also significantly increased liver weight, suggesting an adverse effect on the liver. Pharmacological test example 3 Cholesterol lowering effect (2) Test drug suspended in 0.5% CMC in ddY mice was injected with Triton WR1 339 400 mg / kg 1 hour before and 5 hours after intravenous administration rear, Oral administration was performed three times at 20 hours after administration, and 24 hours after administration of Triton WR 1339, decapitation and blood sampling were performed to separate serum. Serum total cholesterol and triglyceride were determined using cholesterol C-test および and triglyceride G-test ヮ. Table 4 shows the results. Table 4 Effect of MK 6059 substance on serum lipids of Triton hyperlipidemia mice Serum lipid (mg%)

Treatment TG / ch contrast 2551.9 Sat 188.4 563.7 ± 25.5

M 6059 100mg / kg.p.o., Tid 2310.1 Sat. 144.3 416.8 Sat. 12.0 ** Nicotinic acid 1000mg / kg. Ρ.0., Tid 1610.4 Sat. 342.9 '438.6 Sat. 35.4 *

N = 6, * P <0.05 ** P <0.01 For Mice with hyperlipidemia treated with Triton, the group receiving 10 OmgZ kg of MK6059 substance was significantly more serum than the group receiving 1000 mg / kg of nicotinic acid. Cholesterol in it was reduced. Pharmacological test example 4 Lipid-lowering effect on cholesterol-fed nomsters A hamster weighing around 140 g (7 animals per group) was loaded with food containing 1% cholesterol and 0.5% cholic acid, and simultaneously suspended on 0.5% CMC. The cloudy test drug was orally administered once a day for 100 days to 100 g body weight per lm for 10 days.

Four hours after the end of the administration on day 10, blood was collected from the abdominal vena cava and used as a measurement sample. Serum total cholesterol (Tch) was quantified using a cholesterol C-Test Co. (Wako Pure Chemical Industries) and triglyceride (TG) using a triglyceride G-Test Co. (Wako Pure Chemical Industries) kit. . The drug dose is 1 Omg / kg, 3 Omg / k _N 10 Omg / k cholestira for MK 6005 substance Min was 400 mg / kg and 80 mg / kg, and the control group received 0.5% CMC. Table 5 shows the results. Table 5 Effect of MK 6059 substance on cholesterol-loaded hamster serum lipids Serum lipids (mg / dl)

Treatment Tch TG Normal animal 146.9 ± 4.3 "220.0 ± 25.5 Control (0.5% chol-diet) 277.3 ± 11.0 270.3 ± 18.0

MK 6059 10mg / kg p.o.268.3 ± 10.3 299.8 ± 28.3

30mg / kg p.o.228.5 ± 3.5 "275.0 ± 17.0 lOOnig / kg p.o.169.6 ± 6.3 ** 259.3 ± 13.5 Cholestyramine 400mg / kg p.o.184.9 ± 4.5" 205.9 ± 26.3

800mg / kg p.o.204.1 ± 13.9 "196.5 ± 31.6

"* p <0.01

For the MK 6059 substance, administration of 30 mg Zkg and 100 mg kg significantly reduced the total cholesterol level as compared to the control group. For cholestyramine, administration of 400 mg / kg also significantly reduced the total cholesterol level compared to the control group, but required a higher dose than the MK6059 substance. Cholesterol absorption inhibition test in 5 pharmacological test examples As shown in the above pharmacological test results, the MK 6059 substance has a marked cholesterol lowering effect in various animals, and the point of action of this pharmacological action should be clarified. In addition, a cholesterol absorption inhibition test was performed in rats.

A test substance was suspended in 0.5% CMC in a rat (about 6 animals per group) weighing approximately 240 g, which had been preliminarily reared on a diet containing 1% cholesterol. It was orally administered once daily in the evening for 10 days. After the continuous oral administration for 10 days, the test was started 1 day after fasting. 1 The test substance was administered to the rat on the 1st day, and then 30 minutes later, [ ³ H] -cholesterol (Chemical No. 1) was used as a tracer. One 0.5 ml mouse was given a single intravenous injection in the tail vein, and 2 mI [ ¹⁴ C] -cholesterol (a first chemical) was given a single oral gavage in the stomach. Plasma and liver were collected 24 hours and 48 hours after tracer administration, and radioactivity was measured. Using the Zilversmit dual isotope ratio method (Proc. Soc. Med., 140, 862-865, 1972) based on the plasma radiation concentration to determine the absorption rate of cholesterol from the intestinal tract, The pharmacological effect on absorption was investigated. The drug dose was 1 Omg / kg for the MK 6059 substance, 3 Omg / k and 10 OmgZkg for the control group, and 0.5% CMC was administered to the control group. The results are shown in Tables 6 and 7. Table 6 Effect of MK 6059 substance on intestinal cholesterol absorption of cholesterol Cholesterol absorption rate (%) Treatment 24 hours 48 hours Control 56.9 ± 4.9 68.4 ± 9.5

MK 6059 10mg / kg 31.4 ± 5.4 "* 36.1 ± 6.5 '

30mg / kg 26.8 ± 7, ** 28.7 ± 8.9 "lOOmg / kg 26.7 ± 2.4 *** 25.7 ± 2.5 '

* · * Ρ <0.001 ** Ρ <0.01 Of MK 6059 substance on uptake of tracer into rat liver Tracer recovery (%)

24 hours later

Treatment [ ³ H] -cholesterol [ ¹ C] -cholesterol Control 37.0 ± 2.2 25.9 ± 2.0

MK 6059 lOmg / kg 35.2 ± 3.4 13.3 ± 2.8

30mg / kg 35.3 ± 2.5 9.9 ± 2.1

lOOmg / kg 30.0 ± 2.9 7.7 ± 0.3

48 hours later

Treatment [ ³ H] -cholesterol [C] -cholesterol Control 32.7 ± 6.9 27.0 ± 6.7

MK 6059 lOmg / kg 34.4 ± 2.8 14.4 ± 3.4

30mg / kg 33.2 ± 4.9 10.6 ± 4.0

lOOmg / kg 28.3 ± 5.4 7.4 ± 2.1

As is clear from Table 6, the MK6059 substance showed a remarkable inhibitory effect on cholesterol absorption even at the lowest dosage of 1 OmgZkg. In addition, as is clear from the results of measurement of the uptake of the intravenously administered tracer into the liver and the uptake of the orally administered tracer into the liver (Table 7), almost all of the intravenously administered tracer was observed. The uptake rate of the orally administered tracer into the liver was about 50% or less of that in the control group, whereas the uptake was in the liver. This suggests that the cholesterol-lowering activity of the MK6059 substance is due to inhibition of intestinal cholesterol absorption.

Except for resin, lipid-lowering drugs based on cholesterol absorption inhibition have already been There are no existing drugs, and the MK 659 substance is useful as a new type of lipid lowering agent. As shown in the above pharmacological test examples, the MK609 substance has an effect of lowering serum cholesterol or triglyceride, and is expected to be applied as a therapeutic drug for hyperlipidemia. INDUSTRIAL APPLICABILITY The MK609 substance of the present invention is expected to be used as an antifungal agent and a hyperlipidemic agent as shown in Examples.

Claims

Claims MK 6509 substance _{c represented by the following} formula (I)

2. An MK characterized by culturing a microorganism belonging to the micrococcal bacillus and having the ability to produce the MK609 substance according to claim 1, and collecting the MK609 substance from the culture. 6 0 5 9 Production method of substances.

3. The method according to claim 2, wherein the micrococcal bacillus is a microorganism belonging to the genus Elysidosis.

4. The production method according to claim 3, wherein the microorganism belonging to the genus Elysidosis is Elysidosis.

5. A pharmaceutical composition comprising the MK 6509 substance according to claim 1 as an active ingredient.

6. The pharmaceutical composition according to claim 5, which is an antifungal agent.

7. The pharmaceutical composition according to claim 5, which is a therapeutic agent for hyperlipidemia.