JP2001354695A - New physiologically active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new physiologically active substance having an activity for inhibiting a fungal attachment. SOLUTION: This physiologically active substance is obtained by isolating a compound expressed by formula (I) from a culturing liquid of a bacterial strain belonging to the genus Mortierella, and analyzing its structure.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an agent which inhibits fungal adhesion and inhibits fungi from exhibiting pathogenicity.

[0002]

2. Description of the Related Art In recent years, countermeasures against opportunistic infections have become more and more important due to an increase in the number of patients and elderly people whose immune function has decreased due to advanced chemotherapy and the like. Visceral fungal infections caused by Candida, Aspergillus, Cryptococcus, etc. account for some of these opportunistic infections, and the incidence is increasing year by year, and despite being a direct cause of death,
Few effective treatments. Until now, therapeutic agents for fungal infectious diseases have mainly been strategies for chemically modifying known skeletons to develop new compounds. However, due to the problem of resistant bacteria, development of new drugs based on new mechanisms has been eagerly desired. As one of the most promising approaches, attempts to prevent the pathogen from exerting its pathogenicity by inhibiting the attachment of the pathogen to the host have been attracting attention. Recent studies have also reported several relationships between adhesion and infection (Janet F et al., Science, 283:
1535-1538 (1999), Ed T. Buurman et al., Proc. Nat.
l. Acad. Sci. USA, 95: 7670-7675 (1998), Cheryl A.
Gale et al., Science, 279: 1355-1358 (1998)).

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a therapeutic agent for fungal infectious diseases by isolating a novel substance which inhibits the attachment of fungi to cells and inhibits fungal pathogenesis.

[0004]

Means for Solving the Problems In view of the above-mentioned situation, the present inventors conducted a screening for a substance which inhibits the attachment of fungi to cells using a microorganism culture solution as a raw material. As a result, they have found that a substance that inhibits the attachment of fungi to cells is produced in a culture solution of a microorganism belonging to the genus Mortierella. Isolation and structure determination of this active substance revealed that it was a novel active substance. That is, the present invention provides: (1) A compound represented by the formula (I).

Embedded image (2). An antifungal agent comprising the compound according to (1). (3). An antifungal agent comprising the compound according to (1), which inhibits fungal adhesion. (4). Mortierella alpina Mer-f2368, FERM P-17653
Is cultured in a nutrient medium, and the compound described in (1) is collected from the culture broth.

[0005]

DETAILED DESCRIPTION OF THE INVENTION 1. Screening method for producing bacteria 2. properties of the isolated production bacteria; 3. Culture method of production bacteria; 4. Purification method of active substance; The use of the active substance as an antifungal agent will be described in detail in order.

[0006] 1. Screening Method for Producing Bacteria Mammalian cells, preferably intestinal epithelial cells to which fungi adhere, are cultured and fixed by an appropriate method, for example, ethanol. The test sample is inoculated with Candida albicans incubated for an appropriate period of time, cultured for a certain period of time, washed with a buffer, and overlaid with an agar medium, for example, Sabouraud dextrose agar medium (Difco). Three
After overnight culture at 0 ° C., count the CFU and calculate the adhesion rate.

[0007] 2. It is expected that any of the species of Mortierella can be used as a raw material for purifying the compound of the present invention as a purification raw material of the compound of the present invention.However, as a representative strain of the present invention, from the soil collected in Kanagawa Prefecture, In the isolated strain, the present inventors
(Mer-f2368). The bacteriological properties of this MIFR 2368 strain were as follows. Culture was performed at 25 ° C. The medium used was a potato dextrose agar medium. The vegetative mycelium of this strain developed well and formed aerial mycelia. Spore stalks produced from aerial mycelium did not branch, were about 80 μm in length, and tended to taper toward the tip. The sporangia had a sub-spherical shape with a diameter of 15 μm, and the sporangium was smooth and released spores by lysis. The spores were smooth, colorless single cells, showing a renal shape from an oval shape, and the size was 2.5-4 × 1.5-2 μm. Based on the above morphological characteristics, this bacterium is Mortierella alpina
Species identified. The present inventors have proposed that the bacterium is Mortierella alpina MFR 2368 (Mortierel
la alpina Mer-f2368) and deposited with the National Institute of Advanced Industrial Science and Technology under the number FERM P-17653.

[0008] 3. Culture of Produced Bacteria The culture of the above microorganisms is preferably carried out under aerobic conditions, such as a shaking culture by liquid culture and an aeration-agitation culture. The medium used for the culture may be any medium containing a nutrient source that can be used by microorganisms belonging to the genus Mortierella, and any of various synthetic media, semi-synthetic media, natural media, and the like can be used. As a medium composition, a carbon source such as glucose, sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, and organic acids can be used alone or in combination. Pharmamedia as the nitrogen source,
Peptone, meat extract, yeast extract, soy flour, casein,
Organic nitrogen sources such as amino acids and urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate may be used alone or in combination. Sodium salts, potassium salts, magnesium salts, phosphates, other heavy metal salts and the like can be added and used as necessary. Incidentally, when the foaming during the culture is remarkable, various known antifoaming agents can be appropriately added to the medium,
The addition must not adversely affect the production of the target substance. The pH of the medium is desirably in the optimum pH range of the microorganism, usually around neutrality. The culture temperature is a temperature at which the microorganisms grow well, usually 20 to 40 ° C, and particularly preferably 25 ° C.
It is better to keep it near. The culture time is 1 to 1 for liquid culture.
It is about four days. The various culture conditions described above can be appropriately changed according to the type and characteristics of the microorganism used, external conditions, and the like, and the optimal conditions are selected and adjusted from the above range according to each.

4. Purification Method of Active Substance After completion of the culture, in order to collect compound EM-f2368 from the culture solution, a separation and purification method generally used for isolating a microbial metabolite from the culture solution can be used. For example, organic solvent extraction using butanol, ethyl acetate, chloroform, etc., various ion exchange chromatography,
Any known method such as gel filtration chromatography using Sephadex LH-20, adsorption chromatography using activated carbon, silica gel, or the like, or adsorption / desorption treatment using thin-layer chromatography, or high-performance liquid chromatography using a reversed-phase column or the like The method corresponds to this. Further, the present invention is not particularly limited to the method shown here.
Combine these methods alone or in any order,
In addition, compound EM-f2368 can be isolated and collected by repeated use.

[0010] 5. Use of the Active Substances as Antifungal Agents The isolated compounds had fungal adhesion inhibitory activity. Therefore, it is envisaged that the compound is used firstly for prophylactic administration to patients with a high risk factor for fungal infections, and secondly, in combination with existing antifungal agents for refractory fungal infections. When the compound is administered as a therapeutic or prophylactic agent for various diseases, it may be orally administered as tablets, powders, granules, capsules, syrups and the like, or as sprays, suppositories, injections, and external preparations. It may also be administered parenterally as drops.
The dosage varies significantly depending on the severity of symptoms, age, type of liver disease, etc., but is usually about 1 mg to 100 mg per adult per day.
It is administered once or several times a day. At the time of formulation, it is manufactured by an ordinary method using a usual formulation carrier. That is, when preparing a solid preparation for oral use, an excipient, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, etc. are added to the main drug, if necessary, and then a tablet, Coated tablets, granules, powders, capsules, etc. These tablets and granules may of course be sugar-coated, gelatin-coated or otherwise coated as needed. When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, and the like are added to the main drug, if necessary, to give a subcutaneous, intramuscular, or intravenous injection according to a conventional method.

[0011]

According to the present invention, it has become possible to suppress the adhesion of fungi and reduce the pathogenicity.

[0012]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[Example 1] Purification method Mortierella alpina MFR 2368
From a slant medium (potato dextrose agar medium) of the strain (Mortierella alpinaMer-f2368), one platinum loop was added to a 20 ml seed medium (potato starch 2%, glucose 1%, soybean flour 2%,
Potassium monophosphate 0.1%, magnesium sulfate heptahydrate
The mixture was inoculated into a 250 ml Erlenmeyer flask containing 0.05%, pH not adjusted) and cultured on a rotary stirrer at 25 ° C. for 3 days to obtain a seed culture. 1 ml of this seed culture is mixed with 60 ml of medium having the same composition as above.
Was inoculated into a 500 ml Erlenmeyer flask containing, and cultured at 25 ° C for 96 hours on a rotary stirrer. After completion of the culture, 3 L of the obtained culture solution was extracted with 3 L of n-butanol. N- obtained
The butanol extract was concentrated under reduced pressure to obtain 3.0 g of a crude extract. This crude extract was dissolved in a small amount of dimethyl sulfoxide, and the solution was subjected to ODS column chromatography (YMC-GEL ODS-AM 12
0-S50 (manufactured by YMC), inner diameter 50 mm, length 500 mm). Washing with 30% acetonitrile was followed by elution with 50% acetonitrile. The eluate was analyzed by high performance liquid chromatograph, and EM-f2368 (column: J'sphere ODS-H8
0, inner diameter 4.6mm, length 75mm, manufactured by YMC, moving bed:
Acetonitrile-0.01% TFA = 30: 70, flow rate: 1 ml / min,
Detection: eluate containing ultraviolet absorption at 210 nm, retention time: 7.6 min) was collected and concentrated to dryness under reduced pressure to obtain a crude extract of EM-f2368. The obtained crude extract was dissolved in a small amount of dimethyl sulfoxide, and the solution was separated by preparative high performance liquid chromatography (column: J'sphere ODS-H80, inner diameter 20 mm, length 250 mm, manufactured by YMC, moving layer: acetonitrile-0.01% TFA) = 3
0:70, flow rate: 10 ml / min, detection: ultraviolet absorption at 210 nm, retention time: 30.0 min). This fractionation was repeated, and the eluates containing EM-f2368 were combined and concentrated to dryness under reduced pressure to obtain 10.0 mg of pure white powder of EM-f2368.

[Example 2] Physicochemical properties of EM-f2368 1. Color and properties: white powder Molecular _{formula: C 43 H 60 N 10 O} 8 3. Molecular weight: 844 4. UV absorption spectrum: λ _max (MeOH, nm (ε)): 280
(1400) 5. Infrared absorption spectrum: (KBr, cm- ¹ ) 3260, 1680,
1540, 1440, 1400, 1210, 1140, 840, 800, 730 ¹ H nuclear magnetic resonance spectrum _{(600MHz, DMSO-d 6)} , δp
pm: 10.73 (1H, bs), 9.11 (1H, s), 8.43 (1H, bs), 7.97 (1H, d,
7.4), 7.91 (1H, bs), 7.86 (1H, d, 8.5), 7.72 (1H, bs), 7.52 (1
H, d, 7.4), 7.30 (1H, d, 8.2), 7.26 (2H, dd, 7.4,6.6), 7.23 (2
H, d, 7.4), 7.18 (1H, dd, 6.6,6.6), 7.13 (1H, bs), 7.03 (1H, d
d, 8.2,7.4), 6.94 (1H, dd, 7.4,7.4), 6.54 (1H, bs), 6.43 (1
H, bs), 4.62 (1H, m), 4.34 (1H, bs), 4.24 (1H, m), 4.20 (1H, m
m), 4.11 (1H, m), 3.21 (1H, dd, 15.2,5.0), 3.15 (1H, m), 3.04
(1H, m), 2.99 (1H, m), 2.77 (1H, m), 1.91 (1H, m), 1.84 (3H,
s), 1.64 (1H, m), 1.53 (3H, d, 6.9), 1.50 (1H, m), 1.40 (2H,
m), 1.22 (1H, m), 1.14 (2H, m), 0.81 (3H, d, 6.5), 0.80 (3H, d,
6.5), 0.73 (3H, d, 6.3), 0.70 (3H, d, 6.3) 7. Solubility: Soluble: dimethyl sulfoxide Insoluble: hexane, ethyl acetate High performance liquid chromatography analysis conditions: Column: J'sphere ODS-H80, inner diameter 4.6 mm, length 75 mm (manufactured by YMC) Solvent: acetonitrile-0.01% TFA = 30:70 Flow rate: 1 ml / min Detection: ultraviolet at 210 nm Absorption retention time: 7.6min

Example 3 Fungal adhesion inhibitory activity of f2368 1 × 10 ^{5 in} D-MEM medium (Nissui Pharmaceutical) containing 10% fetal bovine serum and 2 mM glutamine in each well of a 6-well multiwell plate
IEC-18 cells adjusted to the number of cells / ml were dispensed at 3 ml each. After the plate was cultured in a carbon dioxide incubator at 37 ° C. for 3 days, the culture supernatant was removed and fixed with ethanol. Of each concentration
f2368-containing Sabouraud dextrose liquid medium at 30 ° C, 4
Candida albicans cultured for 8 hours was adjusted to 4 × 10 ² cells / ml, and 1 ml was inoculated into each well of the plate. After culturing at 30 ° C for 1 hour, remove the culture supernatant, wash with PBS,
2 ml of dextrose agar medium (Difco) was overlaid. 30
After culturing overnight at ℃, CFU was counted and the adhesion rate was calculated. The result is shown in FIG. f2368 showed an activity of inhibiting the attachment of Candida albicans to IEC-18 cells.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the concentration of f2368 and the rate of Candida adhesion.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1: 645) A61K 37/02 (72) Inventor Noriaki Sakata 3-14 Sanrakucho, Yatsushiro-shi, Kumamoto (72) Invention Person Kazuyuki Dobashi 1-3-5, Minamigaoka, Hadano-shi, Kanagawa Prefecture 3-204 F term (reference) 4B064 AG01 BA09 BE19 BG01 BG09 BH02 BH04 BH06 CA05 DA03 4C084 AA02 AA06 AA07 BA01 BA10 BA17 CA05 DA42 NA14 ZB352 4H045A10A EA29 FA72 GA01 GA22 HA02

Claims (4)

[Claims] 1. A compound represented by the formula (I). Embedded image 2. An antifungal agent comprising the compound according to claim 1. 3. An antifungal agent comprising the compound according to claim 1 and inhibiting fungal adhesion. 4. Mortierella alpina. Mer-f2368, FERM
The method for producing a compound according to claim 1, wherein P-17653) is cultured in a nutrient medium, and the compound according to claim 1 is collected from the culture solution.