JPS62270596A - Infection-preventing substance aladapcin - Google Patents

Abstract

NEW MATERIAL:Aladapcin having the following physical and chemical properties. Properties of the substance: ampholytic water-soluble white powder. Molecular formula: C13H25N5O5. Molecular weight: 331. Solubility: soluble in water and methanol and insoluble in acetone, ethyl acetate and chloroform. Color reaction: positive in ninhydrin and peptide reagent. Acid hydrolysis: 1mol meso- diaminopimelic acid and 2mol D-alanine are detected after hydrolyzed with 6NHCl at 105 deg.C for 20hr. USE:An enhancer having host resistance targeting various microbisms. PREPARATION:A bacterium [preferably Nocardia sp. SANK 60484 strain (FERM P-7432)] belonging to the genus Nocardia, capable of producing aladapcin, is cultivated preferably in a liquid medium by shaking culture or aerated spinner culture at about 28 deg.C for 80-96hr.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention The present invention utilizes a novel infection-preventing substance, alladapsin (Aladapsin).
pcin) and its manufacturing method. The present inventors have demonstrated that the 5ANK 60484 strain belonging to the genus Nocalzoa, which was isolated from soil collected in Shioya District, Tochigi Prefecture, produces a novel infection-preventing substance that has an infection-preventing effect against infection by Durham-positive and Durham-negative #I bacteria. This was discovered and was named Aladapsin. Therefore, alladapsin is used for the prevention or treatment of human and animal diseases caused by these bacteria. 5 which produces alladapsin
The mycological properties of ANK60484 strain are as follows.

1) Morphological characteristics 1113P [International Streptomyces
Project (International St, r
eptomycesProject)] defined medium and Waxman (S, A.

Waksman et al. recommended that the actin mycetes (
'I'l)s Actinomycetes), 2
Culture on the medium of Volume]. Cultivation is usually carried out at 28°C. S
The morphological characteristics of AN'I (strain 60484) are that aerial hyphae do not occur, vegetative hyphae are well elongated, and zigzag (Zig-Z) is observed.
ag) and a few days after potash inoculation culture, clear divisions are observed in the vegetative hyphae. The segmented hyphae exhibit spherical to pear-shaped shapes of various sizes. In addition, the formation of special organs is not observed.

2) Properties on various culture media Properties when cultured on various culture media at 28°C for 14 days are as shown in Table 1. The color tone display represents the color chip number of the Japan Color Research Institute version 1 standard color chart.

Table 1 The results after 14 days at 28°C are shown.

Medium Name Properties 4-'1°”1
□ Light yellowish brown (6-8-8) 0"5P2) s
p Production Izua 7″ Ragi 7th R Yellowish gray (1-9-10) (ISP
5) sp Extract without producing iron agar R light yellow tea (6-8-9) (ISP 6) sp
No production medium name Characteristic sp No production asparagine agar R light yellowish orange (2-9-9) sp
No production sp No production Medium name Properties Water agar R yellowing method (1-9-10) sp No production 3) Physiological properties The physiological properties of 5ANK 60484 strain are shown in Table 2, and the carbon source assimilation ability is shown in Table 2. It is shown in Table 3.

Table 2 Physiological properties *Medium 1: Trisiton Yeast Expros (IS
P 1) Medium 2: Peptone, yeast extract, iron agar (
ISI) 6) Medium 3: Tyrosine agar (ISP 7) 4) Regarding intracellular components of M.B. Lecbev
Alier et al.'s method [A.
s tz ) et al., Classification of actinomycetes (Actj, nomy
cete taxonomy), p. 225, 198
As a result of perchromatography analysis of the acid hydrolyzate of the bacterial cells, mesodiaminopimelic acid, arabinose, and galactose were found.
The cell wall type was confirmed to be type ■. The glycotype of all bacterial cells was A type. Furthermore, the method of H. Mordarska et al.
Ono General Microbiology (Journ)
al of general Microbology
), vol. 71, p. 77, 1972], the results showed that LCN-A (Lipids ch.
aracterj, 5tjc of Nocardia
) existed.

Among the above-mentioned properties of the 5ANK 60484 strain, in particular: 1. Mycelia are fragmented, 2. The cell wall is type 2, and the glycotype in all bacterial cells is type A. 2. LCN-A is present in the bacterial cells. Based on these findings, this bacterial strain is a member of the Nocardia family (Nocardl, aceae) among actinomycetes.
1. It is clear that the strain belongs to the genus ocar6 j-a). Nocardia sp.
sp,) SANK 60484 (Feikoken Bacillus No. 742
No. 3).

The bacteria that produce alamezosin have been explained above, but it is well known that the properties of actinomycetes are not constant and can easily change naturally or artificially. It includes all strains that produce alladapsin belonging to the genus.

Cultivation in the present invention is carried out in accordance with the culture method for general actinomycetes, and is preferably carried out by shaking culture or aerated agitation culture in a liquid medium. As a medium component, for example, P-sugar is used as a carbon source.

Maltose, sucrose, mannitol, molasses.

Glycerin, dextrin, starch, soybean oil, cottonseed oil, etc. are used as nitrogen sources, and soybean flour and peanut flour are used as nitrogen sources.

Cottonseed flour, Farmamine, fishmeal, corn steep liquor, pezotone, meat extract, Ease l-.

Yeast extract, sodium nitrate, ammonium nitrate, ammonium sulfate, etc., and inorganic salts such as common salt, phosphate, calcium carbonate, micrometal salts, etc. are added as appropriate. For liquid culture, silicone oil, vegetable oil, and surfactant are appropriately used as antifoaming agents.

The voice of the medium is around 7.0, the culture temperature is 24℃ to 30℃,
Particularly preferred is around 28°C. Usually, the production amount of alladapsin reaches its maximum value after 80 to 96 hours of culture. Alladapsin, which is mainly present in the liquid part of the culture solution, is removed by filtering or centrifugation using soil as a filter aid to remove bacterial cells and other solid parts, and the filtrate or supernatant is removed. Extracted and purified from clear water. Alladapsin can be collected by utilizing its physicochemical properties, for example, using an adsorbent. As an adsorbent, for example, activated carbon or adsorption resin Amberly) XA
D-2.

XAD-4, XA, D-7, etc. (manufactured by Rohm and Haas)
゜CHP20P (manufactured by Mitsubishi Chemical Industries, Ltd.) is used. The solution containing aladapsin is passed through a layer of adsorbent such as the one described above to adsorb and remove impurities, or after adsorbing aladapsin, it is eluted using methanol water, acetone water, n-butanol water, etc. 6 Also,
Methods for collecting neutral fat-soluble substances from culture solutions, such as organic solvents that are immiscible with water, such as chloroform, ethyl acetate,
Aladapsin can also be purified by extracting impurities from the culture solution or aqueous solution using n-sitanol or the like alone or in combination. To further purify alladapsin, partition column chromatography using cellulose such as Avicel (manufactured by Asahi Kasei Industries, Ltd.), Sephadex LH-20 (manufactured by Pharmacia), etc., and reversed phase using a reversed phase carrier are performed. Effective methods include column chromatography, an extraction method that utilizes the difference in the distribution ratio of aladapsin and mixed impurities to the solvent, or a countercurrent distribution method. Use the above purification methods alone! Sialadazosin can be purified by repeatedly using the above methods or by appropriately combining them. Alladapsin, like general lipid-soluble antibiotics, exists in the bacterial cell portion of the culture medium depending on the culture conditions. Therefore, it is possible to extract with a hydrophilic organic solvent such as alcohol or acetone, remove the solvent, make an aqueous solution, and then extract and purify it in the same manner as from the culture filtrate.

Alladapsin thus obtained has the following physicochemical properties.

(1) Properties of the substance: Amphoteric, water-soluble, white powder.

(2) Molecular formula: C43H25N5O5 (3) Molecular weight: 331 (4) Solubility: Soluble in water and methanol, insoluble in acetone, ethyl acetate, and chloroform.

(5) Color reaction: Positive for ninhydrin and peptide reagents.

(6) Thin layer chromatography; 0.42 Adsorbent; Eastman Chromagram Sheet A132
54 developing solvent; n-butanol:water:acetic acid (4:1:2
) (7) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in an aqueous solution is 220 nm.
No specific absorption is shown above, only terminal absorption is shown.

(8) Infrared absorption spectrum = νgH, "The infrared absorption spectrum measured by the IKBr method is as shown in FIG.

(9) Nuclear magnetic resonance absorption spectrum: Nuclear magnetic resonance absorption spectrum (270MHz) measured in δ' ppm heavy water using dry MS (tetramethylsilane) as an external standard.
is as shown in FIG.

α1 liquid chromatography: Column: Microbondapak Cl8 (manufactured by Waters) Solvent: 5mM sodium heptane sulfonate-1
, 5 Thiacetonitrile flow rate: 1°5 p/min Uv: 210 nm Holding time: 10.8 min α-acid hydrolysis: 6 As a result of hydrolysis at 105°C for 20 hours with NHCt,
1 mole of meso-diaminopimelic acid and 2 moles of D-alanine were detected.

0bori infection prevention effect Alladapsin was administered to mice 24 hours prior to S,C”.

After administration, L coli 5ANK 73175 (2X
1.V. infection was performed using 10' cells Al), and the number of survivors was examined after 7 days.

From the above, alladapsin is used alone or in combination with a commercially available antibacterial agent as a host resistance enhancer for various bacterial infectious diseases. Its administration forms include subcutaneous injection,
Parenteral administration methods include intravenous injection, intramuscular injection, and suppositories, and oral administration methods include tablets, capsules, powders, and granules. The dosage varies depending on the target disease, route of administration, frequency of administration, etc., but for adults, it is usually preferable to administer 10 to 1 g per day, either once or in divided doses.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 One platinum loop of Nocardia sp. 5ANK 60484 strain was inoculated into a 21-volume Erlenmeyer flask containing 500su of the medium shown in medium composition-1, and cultured at 28°C for 96 hours in a rotary shaking incubator at 22 Orpm for 7 h.

This culture solution 2,51 containing the same medium 5O1) 100
Inoculated into a 1-volume tank and heated to 28°C by rotating at 100 rpm.
Culture was carried out with aeration for 48 hours. This culture solution 151 was inoculated into a 6001 volume tank containing the same medium 3001, and the temperature was 2.
8°C/airflow rate 0.5 vvm.

Internal pressure], Okg·cJ, rotation at 100 rpm for 91 hours with aeration and agitation. Obtained culture solution 700
1, add 30 kg of Celite 545 (manufactured by Johns Mampil Products Corporation, USA) as a filter aid.
In addition, culture F solution 6901 was obtained by heating. Adjust the culture p solution to pH 5.5 and add it to Diaion 8-I
B (H+) (manufactured by Mitsubishi Chemical Industries, Ltd.) 100A! column to adsorb alladapsin. After washing with 300 J of deionized water, it was eluted with 0.5N ammonia water 4001. The obtained eluate 1801 was concentrated under reduced pressure to obtain a concentrated solution 81. Adjust concentrate 81 to -6.5 and a7
Bar 5 a) C! G-5O(H+) (ROHM・
& Haas: t! It was adsorbed onto a 47 column.

After washing with 251 deionized water, it was eluted with 0.0!5N hydrochloric acid water.

The obtained eluate 23/ was adjusted to -7.0 with sodium hydroxide and concentrated under reduced pressure to obtain concentrate 21. Avicel (manufactured by Asahi Kasei Corporation) 3001 was added to the obtained concentrate 21.
1 was added and freeze-dried.

The freeze-dried powder was pre-mixed with 80% thiacetonitrile-water (
Layered on 41 Avicel columns packed with V/V)
8 (l acetonitrile-water, 201 ml of 70% acetonitrile-water, 241 ml of 20% acetonitrile-water. 70% and 20% acetonitrile-water.
When fractionation was carried out in units of A', the active substance was eluted in fractions 4 to 6, which were eluted with 201 acetonitrile-water.

These fractions were collected, concentrated under reduced pressure, and freeze-dried to obtain a crude powder of 1
．． 91 was obtained. 1.92 g of the obtained coarse powder was added to 8
0% acetonate) was adsorbed onto a 25OWL Avicel column packed with IJ Roux water, and 1.51% of 70%, 5% of each
O1% and 30% acetonitrile-5O01lI in water
It was eluted sequentially in t increments.

The active substance was eluted in fractions A6-9, and these fractions were collected, concentrated under reduced pressure, and freeze-dried to give a powder of 354.8
W was obtained. The obtained powder (354.8 cm) was adsorbed on a column of 270 wLl of Amberlite CG-5O (NH4''), developed with water, and fractionated into 51 fractions.

Fractions A38 to A60 containing alladapsin were collected and lyophilized to obtain 13.7 cm of white powder.

Medium composition - 1 Glucose 5% Soy flour 0.75 Ammonium sulfate 05 Proflo 0.6 Ca Co 3 (155 KH2P0401 pl (7.0) Toyo Pearl HW-40 adjusted with water (
Fine) (Toyo Soda Kogyo (manufactured by Rikisha)) It was adsorbed on a 30 inch column and developed and eluted with water.

When the eluate was fractionated into 3 ml portions, alladapsin was detected as a spot in the upper half of the thin layer chromatogram in fractions A32-5.
It was eluted by 2. The eluted fractions were collected, concentrated under reduced pressure, and lyophilized to obtain 5.4 cm of alladapsin as a white powder.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of aladapsin,
Figure 2 shows the nuclear magnetic resonance absorption spectrum of the same material.

Claims (1)

[Claims] 1. Alladapsin having the following physical and chemical properties. (1) Properties of substance: Amphoteric, water-soluble, white powder. (2) Molecular formula: C_1_3H_2_5N_5O_5(3
) Molecular weight: 331 (4) Solubility: Soluble in water and methanol, insoluble in acetone, ethyl acetate, and chloroform. (5) Color reaction: Positive for ninhydrin and peptide reagents. (6) Thin layer chromatography; R_f value; 0.42 Adsorbent; Eastman Chromagram Sheet No. 1
3254 developing solvent; n-butanol:water:acetic acid (4:1
:2) (7) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in an aqueous solution shows no specific absorption at 220 nm or more, but only terminal absorption. (8) Infrared absorption spectrum: ν^K^B^r_m_a
The infrared absorption spectrum measured by the _xcm^-^1KBr method is as shown in FIG. (9) Nuclear magnetic resonance absorption spectrum: δ: ppm heavy water,
The nuclear magnetic resonance absorption spectrum (270 MHz) measured using TMS (tetramethylsilane) as an external standard is shown in FIG. (10) Liquid chromatography: Column: Micro Bonder Pack C_1_8 (manufactured by Waters) Solvent: 5mM sodium heptane sulfonate-1.5% acetonitrile Flow rate: 1.5ml/min UV: 210nm Retention time: 10.8 minutes ( 11) Acid hydrolysis: As a result of hydrolysis with 6N HCl at 105°C for 20 hours, 1 mol of meso-diaminopimelic acid and 2 mol of D-alanine were detected. 2. A method for producing alladapsin, which comprises culturing alladapsin-producing bacteria belonging to the genus Nocardia and collecting alladapsin from the culture solution. 3. The production method according to claim 2, wherein the alladapsin-producing bacterium belonging to the genus Nocardia is Nocardia sp.