JPS6344128B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a protective agent against infectious diseases containing sulfated polysaccharide DF-4639 as an active ingredient. Sulfated polysaccharide DF-4639 is produced by DF-4639-producing bacteria belonging to Arthrobacter (for example, tentatively named Micrococcus AT-25).
Also, Arthrobacter AT-25
to the Institute of Microbial Technology under the name of
It is produced by culturing the bacteria deposited as FERM P-5255 and FERM BP-1357. The mycological properties of this AT-25 bacterium are as follows. (a) Morphology: Spherical to short rod-shaped (spherical at late stage of culture) 0.5~
0.7×0.7-1.5μm polymorphism, no motility,
No sporulation, positive Gram staining, no acid-fastness (b) Growth status in each medium Broth agar plate culture Moderate dot-like or circular growth, shiny bright brown color, but non-diffusive. Juicy agar slant culture The growth was similar to that of the plate culture except that it grew in a filamentous manner. Meat juice agar liquid culture No bacterial film formation, turbidity and sediment throughout. Meat juice gelatin puncture culture (culture at 22°C) Grows along the puncture line and liquefies into a turnip. Litmus milk medium Turns litmus slightly red. Coagulation is also observed in long-term culture (14 days or more). (c) Physiological properties Nitrate reduction negative, denitrification reaction negative,
MR reaction negative, VP reaction negative, indole formation none, hydrogen sulfide generation none, starch hydrolysis ability none, citric acid utilization none, inorganic nitrogen source utilization yes, water-insoluble pigment formation yes, urease action none , no oxidase effect,
Catalase action, growth range PH6.0
~8.5 (optimum 7~8), temperature 10~37℃ (optimum 27~
30℃), aerobic, OF reaction negative, below 15
No acid and gas production from seed sugars (1) L-arabinose, (2) D-xylose, (3) D-glucose, (4) D-mannose, (5) D-fructose, (6) D -galactose, (7) maltose, (8) sucrose, (9) lactose, (10) trehalose, (11) D-sorbitol, (12) D-mannitol, (13) inosite, (14) glycerin, (15) ) Starch (d) Other properties Phosphatase action None, ability to decompose arginine None, grows in 7.5% sodium chloride but not 10%, minimum inhibitory concentration for novobiocin and lysozyme are both 3.1 μg/ml, Guanine and cytosine content (GC content) 72.9%, intracellular pigment (yellow) production, cell wall component contains diaminopimelic acid (DAP).
Considering the description in the edition, it is appropriate to classify this bacterium as belonging to Arthrobacter. Next, the basic method for obtaining sulfated polysaccharide DF-4639 will be described. That is, DF−4639
In order to obtain DF-4639, it is sufficient to accumulate DF-4639 in the culture solution and the bacterial cells of the bacteria used, and for this purpose, any medium that can grow the bacteria used can be used. In addition to assimilable carbon sources and nitrogen sources, inorganic salts and vitamins can be used alone or in combination in appropriate proportions. Arthrobacter AT-25 is suitable as the bacterium used. Carbon sources include glucose and glycerin, nitrogen sources include yeast extract, meat extract, peptone, soybean flour, cornstarch liquor, and inorganic ammonium salts such as ammonium hydrogen phosphate and ammonium nitrate.Other inorganic salts include magnesium, Appropriate amounts of metal salts such as potassium, iron, manganese, etc. can be added. Also,
Since DF-4639 is a sulfated polysaccharide, it is extremely useful to add an appropriate amount of sulfate such as sodium sulfate or ammonium sulfate. Next, the culture temperature and pH of the medium may be within a range in which the bacteria used can grow, but it is preferable to culture at a temperature of 25 to 37°C and a pH of 6.5 to 8.5. Cultivation is preferably carried out under aerobic conditions, for example by shaking culture or aerated agitation culture in a culture tank. The culture time may be 24 hours or more, but preferably 50 to 200 hours. When the culture is completed, the culture is separated into bacterial cells and a filtrate using a centrifuge, and the filtrate is treated as described below. Regarding bacterial cells,
If the amount is small, it can be ignored, but if it is large, it will be processed separately according to the method described later. That is, a solution of a quaternary ammonium salt, such as cetylpyridinium chloride (CPC), is added to the filtrate and stirred, and the resulting precipitate is separated and collected using a high-speed centrifuge. An electrolyte solution of an appropriate concentration, for example, 3 molar saline containing 10% ethanol, is added to the obtained precipitate and dissolved with stirring. Ethanol is added to the resulting solution until no new precipitate is formed, and the resulting precipitate is collected, washed with ethanol and acetone, and then dried to obtain a coarse powder. The coarse powder obtained here is dissolved in water again,
Add dilute hydrochloric acid under ice cooling to bring the pH to about 1.0, and then centrifuge at low temperature (about 5°C) to remove insoluble matter. Collect the supernatant, neutralize it with dilute caustic soda solution, and add
An aqueous solution of a class ammonium salt, such as cetyltrimethylammonium bromide (CTAB), is added and the resulting precipitate is collected by centrifugation. The obtained precipitate is added to 1 molar saline solution, and impurities such as nucleic acids are thoroughly solubilized by stirring, followed by centrifugation to collect insoluble matter. This insoluble matter is added to 3 molar saline containing 10% ethanol and dissolved while heating to about 50°C.
Ethanol was added to this solution to precipitate it, and the resulting precipitate was collected and washed with ethanol and then acetone, then dissolved in water again with stirring at room temperature, and centrifuged at about 5°C to remove trace amounts of insoluble matter. do. Ethanol was added to the supernatant until no new precipitate was formed, the resulting precipitate was collected, washed sequentially with ethanol, acetone, and ether, and then heated at approximately 70°C.
When dried under reduced pressure, DF-4639 is obtained as a white powder. On the other hand, to obtain DF-4639 from bacterial cells, toluene or the like is added to an aqueous suspension of the bacterial cells, and the suspension is left at room temperature to allow the bacterial cells to self-lyse, and the components within the bacterial cells are transferred to water. Ethanol is added to this suspension until the concentration reaches 40%, precipitated proteins, nucleic acids, etc. are removed by filtration along with bacterial cell residue, and the filtrate is concentrated to remove the ethanol. Add the CPC solution to this, collect the resulting precipitate, and treat it in the same way as the culture filtrate. DF-4639 obtained by the method described above is
It is obtained as a sodium salt of sulfated polysaccharide and has various physicochemical properties as described below. (a) Elemental analysis values, sugar and protein content Table 1 shows the values of 5 lots and the average value.

[Table] (b) Molar ratio of glucose, galactose, sulfate, and phosphorus content DF-4639 was hydrolyzed in 1N sulfuric acid at 100℃ for 5 hours, then neutralized with barium carbonate to form Dowex50W.
(H type) using the effluent obtained by applying
The method of Khym et al. (JXKhym and LPZill, J.
Am.Chem.Soc., 74 , 2090 (1952)) was applied to separate and quantify glucose and galactose. Furthermore, the molar ratio of sulfate groups and phosphorus was calculated from the S and P contents (%). Table 2 shows glucose
An example of the molar ratio of each component when it is 1.0 mol is shown.

[Table] Note that P is present in DF-4639 as glycerol phosphate, glucose phosphate, and muramic acid phosphate. (c) Content molar ratio of amino acids and amino sugars An example of the results obtained by hydrolyzing DF-4639 in 3N hydrochloric acid at 100℃ for 16 hours, distilling off the hydrochloric acid, and quantifying it using an amino acid analyzer, assuming that glutamic acid is 1.0 mol. It is shown in Table 3.

[Table] (d) Optical rotation [α] ²⁵ _D -36±1° (C=1.0, water) (e) Molecular weight 23000 (main peak in gel filtration method) Figure 1 shows the gel filtration diagram (Toyo Soda Co., Ltd.)
Using G4000SW column, solvent 0.1M saline, flow rate
1.1 ml/min, standards Dextran T-500, Dextran T-10 and glucose). (f) Ultraviolet absorption No maximum absorption is observed in the 220-340 nm range in a 2 mg/ml aqueous solution. (g) Infrared absorption spectrum (KBr tablet) As shown in Figure 1, 1240, 840 (shoulder) and 810
It shows an absorption characteristic of sulfated polysaccharide in cm ^-1 . The structure of DF-4639 is estimated to be a sulfated polysaccharide in which a peptidoglycan moiety is bonded to a carbohydrate moiety mainly consisting of D-glucose and D-galactose via muramic acid phosphate. The biological properties of DF-4639 are shown below. (a) In vitro biological activity

[Table] (b) Fibrinolysis induction activity in rats Using rats, the dissolution time (ELT) of euglobulin before and after administration of the sample (intravenous injection) was measured over time, and the rate of increase in fibrinolytic activity (%) was determined by the following formula: Expressed as. Fibrinolytic activity increase rate (%) = FLT (previous value) - FLT (
(later value)/FLT (previous value) x 100

[Table] (c) Acute toxicity LD ₅₀ 1500 mg/Kg or more (mouse, intravenous injection) Next, production examples will be given and explained, and % is W/V % unless otherwise specified. Production example 1 Glucose 2%, peptone 0.5%, cornstarch liquor 0.5%, yeast extract 0.3%, salt 0.5%
Liquid medium 100 consisting of and 0.3% calcium carbonate
1 of Arthrobacter AT-25 bacteria grown on agar slant in advance in a 500 ml shake flask containing 1 ml of Arthrobacter AT-25 bacteria.
A loopful of platinum was inoculated and cultured with shaking at 30°C for 3 days to obtain a seed culture. Next, 2% glycerin, ammonium sulfate
0.5%, monopotassium phosphate 0.1%, sodium sulfate
Medium consisting of 0.05% and yeast extract 0.2% 20 to 30
In addition to the jar fermenter of 200℃ at 120℃
After sterilization for minutes, the pH was adjusted to 7.5. Inoculate this with 600 ml of seed culture solution at a temperature of 30℃ and aeration rate per minute.
10. Cultured for 159 hours under stirring conditions of 250 revolutions per minute. The obtained culture solution was centrifuged to remove a small amount of bacterial cells, and 500 ml of a 10% cetylpyridinium chloride aqueous solution was added to the supernatant liquid 18, and after being left overnight,
The sediment was centrifuged. Add this precipitate to 600 ml of 3M salt-10% (V/V) ethanol solution,
After stirring thoroughly to dissolve, add ethanol 1.6
The resulting precipitate was filtered using a glass filter. This precipitate was washed with ethanol, then with acetone, and dried to obtain 37.0 g of coarse powder. Dissolve this coarse powder in 500ml of water and add 1N at 0°C.
The precipitate produced by adjusting the pH to about 1.0 with hydrochloric acid was removed by centrifugation, the supernatant was neutralized, 500 ml of a 10% aqueous cetyltrimethylammonium bromide solution was added, and the precipitate produced was centrifuged. After thoroughly washing the precipitate with 1M saline, add 150ml of 3M saline-10% (V/V) ethanol solution, stir thoroughly to dissolve, add 450ml of ethanol, leave overnight, and centrifuge to precipitate. I collected things. After washing this precipitate with ethanol, it was dissolved again in 150 ml of water.
Filter with a glass filter and add a small amount of residue to 50ml of water.
Washed with. The filtrate and washing liquid were combined and poured into ethanol 2 with stirring to form a white precipitate. This precipitate was filtered using a glass filter, washed sequentially with ethanol, acetone and ether, and dried under reduced pressure at 55°C for 5 hours to form a white powder.
13.9g of DF-4639 was obtained. This substance exhibited the physical properties described in the text, and the concentration for 50% inhibition of thrombin activity was 0.7 μg/ml. Production Example 2 Add 20 medium consisting of 2% glycerin, 1% soluble starch, 1% meat extract, 1% peptone and 0.2% salt to a 30 volume jar fermenter.
Sterilize at 120°C for 20 minutes, and proceed as in Production Example 1.
Time culture was performed. After the culture was completed, the culture solution was centrifuged and separated into bacterial cells and supernatant. 150 ml of 10% cetylpyridinium chloride aqueous solution was added to the supernatant, and the mixture was separated and purified according to Production Example 1 to obtain 3.2 g of white powder of DF-4639. On the other hand, the bacterial cells were suspended in a mixture of 1200 ml of water and 300 ml of toluene, and left at room temperature for 3 days with occasional stirring. After toluene was distilled off under reduced pressure, ethanol was added to the solution to a concentration of about 40% (V/V), and precipitated proteins, nucleic acids, etc. were removed by filtration along with the bacterial cells. The obtained filtrate is about 1
After concentration under reduced pressure, 30 ml of 10% saline and 100 ml of 10% aqueous cetylpyridinium chloride solution were added, and the resulting precipitate was centrifuged. Below, production example 1
Separate and purify the white powder DF-4639 according to 2.4
I got g. This substance exhibited the physical properties described in the text, and the concentration for 50% inhibition of thrombin activity was 0.8 μg/ml. DF-4639 is effective against bacterial infections caused by bacteria such as Aeruginosa, Escherichia coli, Streptococcus, Staphylococcus, Streptococcus pneumoniae, and Klebsiella pneumoniae, as well as deep fungal infections caused by Candida and the like. Its medicinal properties differ from conventional antibacterial agents and antibiotics, which exhibit bacteriostatic or bactericidal effects by themselves.In particular, it enhances the body's defense function, which primarily increases the number or hyperfunction of neutrophils. It is characterized in that it indirectly suppresses the growth of pathogens in living organisms, particularly in body fluids, and as a result, exerts a protective effect against infections caused by these pathogens. Recent trends in infectious diseases are such that, in addition to simple infections caused by the infiltration of these pathogens, which have been recognized in the past, there is an increase in complex infections accompanied by various serious underlying diseases, which is a serious problem. It's becoming more and more. For example, the cure rate for white blood cells is increasing due to the development of chemotherapeutic agents, but the number of deaths due to concomitant infections such as pneumonia is also increasing. Furthermore, it is a well-known fact that it is extremely difficult to treat granulocytopenia associated with malignant tumors such as white blood cells and malignant lymphoma, and that this is a cause of so-called spontaneous infections. It goes without saying that the infectious diseases of these cachectic patients cannot be completely cured using conventional chemotherapeutic agents alone, and cannot be treated without improving the body's defense function. This drug significantly inhibits granulocytes in animals and humans.
It is particularly effective in improving the quantity and quality of neutrophils, and even this drug alone can be expected to be effective against these complex infections. If this is combined with conventional chemotherapeutic agents, the effect will be further doubled. Experimentally, this drug
By administering 10 μg, it significantly increases the number of granulocytes in mice and guinea pigs and enhances its bactericidal effect. In addition, it has been shown in infection experiments with Escherichia coli that it significantly prevents bacteremia after infection, and is expected to be effective against various infections caused by the various bacteria and fungi mentioned above, including coliform bacteria. Due to the characteristics of this type of drug, the sooner it is administered after the onset of illness, the higher the cure rate will be. The dosage form used includes injections such as intravenous and intramuscular injections, as well as suppositories, tablets, capsules, powders, etc., but injections are most preferred. Dosage ranges from 0.1 to 100
A range of mg/Kg body weight/day is sufficient. Next, an experimental example of the present invention will be shown. Experimental Example 1 A group of 20 STD-ddY male mice (5 weeks old, weight 22-30 g) were used as the animals, and Escherichia coli isolated from a patient with a urinary tract infection was used as the inoculum. Every 24 hours after bacterial inoculation, DF-4639 was dissolved at a rate of 500 μg/ml in 0.01 M, PH7.4 phosphate buffer, and 0.2
ml was administered subcutaneously. For controls, 0.2 ml of the same buffer was administered subcutaneously. For these animals, administer 0.2 ml of a normal bouillon suspension of E. coli (6.0 x 10 ⁷ or 3.0 x 10 ⁷ cells/ml).
was inoculated subcutaneously, and the survival rate was determined from the number of survivors on the 7th day after infection. The experiment was repeated four times under the same conditions, and the results are shown in the table below.

[Table] Experimental Example 2 The number of animals used was 10 per group, and a clinically isolated Cytrobacter bacterium was used as the inoculum solution.
freundii) normal bouillon suspension (2.8×10 ⁸ pieces/
The only difference was that ml) was used, and the rest was exactly the same as Experimental Example 1. The results are shown in the following table.

[Table] Experimental Example 3 The number of animals used was 10 per group, and the inoculum was a suspension of clinically isolated Pseudomonas aeruginosa in normal broth (8.5 x 10 ⁷ cells/ml). The only difference is that the rest is exactly the same as Experimental Example 1. The results are shown in the following table.

[Table] Experimental example 4 Guinea piglet (Hartley strain, female, 5 animals per group)
DF-4639 was subcutaneously administered at 10 μg per animal, and changes in peripheral blood cells over time were measured using blood smears. The results were the values 24 hours before administration (control).
It is shown in the table as an index with 100. The numbers in parentheses are the average number of cells per ^mm3 of blood.

[Table] Experimental Example 5 The antibacterial properties of the following test bacteria were examined using the agar plate dilution method according to the standard method of the Society of Chemotherapy.
The minimum inhibitory concentration (MIC) was 100 mcg/ml or more against any bacteria, and no direct antibacterial activity was observed. Escherichia coli, Citrobacter freundii,
Pseudomonas aeruginosa Schigella flexneri,
Proteus vulgaris, Klebsilla pneumoniae,
Serratia marcescens, Staphylococcus aureus,
Streptococcus pyogenes, Bacillus subtilis

[Brief explanation of the drawing]

FIG. 1 is a gel filtration diagram, and FIG. 2 is an infrared absorption spectrum.

Claims (1)

[Scope of Claims] 1. A preventive agent against infectious diseases containing as an active ingredient DF-4639 having the following physical properties (the physical properties below are for its sodium salt). (1) Molecular weight 23000 (main peak determined by gel filtration method) (2) Elemental analysis values (indicating the width of 5 lots) C26.20-26.90%, H3.77-3.96%, N0.71-1.18%, S10. 7-11.6%, P0.75-1.02% (3) Sugar and protein content Sugar content (%): 56.4 (phenol sulfuric acid method, galactose standard) Protein content (%): 2.0 (Lowrie-Follin method,
Bovine serum albumin standard) (4) Specific rotation [α] ²⁵ _D −36±1° (1.0% aqueous solution) (5) Main absorption band in infrared absorption spectrum (cm ^-1 ; KBr) 1240, 840 (shoulder), 810 (6) Solubility Easily soluble in water, almost insoluble in organic solvents such as ether, benzene, chloroform, methanol, and ethanol. (7) Color reactions Phenol-sulfuric acid, Anthrone-sulfuric acid, Biuretz reaction, and Lowry-Follin reaction are positive, Elson-Morgan reaction and ninhydrin reaction of aqueous solution are also positive, and carbazole reaction and Sakaguchi reaction are negative. (8) Basic, neutral, acidic pH of aqueous solution 6-8 (3% concentration) (9) Content of constituent sugars, sulfate groups, and phosphorus D-glucose, D-galactose,
The molar ratio of SO ₃ Na and P (phosphorus) is approximately 10:63:75:7, respectively, with D-glucose being 10.
It is. (10) Content of constituent amino acids and amino sugars As a result of analysis using an amino acid analyzer, the molar ratio of glutamic acid, alanine, glycine, L,L-diaminopimelic acid, glucosamine, and muramic acid is approximately 1:1 when glutamic acid is 1: The ratio is 2:1:1:2:1.