The way to obtain sulfated derivative galactan

 

(57) Abstract:

Usage: Microbiology, pharmacology. The entity conducting the mild acidic hydrolysis azillion consisting of 80% neutral carbohydrate, 20% lipids and 25% protein, with mol.m. 12500 extracted from bacteria Klebsiella pneumonial. The resulting mixture was separated by chromatography on aminoalkenes, lyophilizer carbohydrate fraction and subjecting it gel filtration. The fraction containing galactan mol.m. 4000 - 10000, lyophilizers and subjected to sulfation. The reaction mixture deleteroute and get a new sulfated derivative galactan with the following features: formed by the linear sequence of residues of galactose connected by the links (1 - 3), contains 20 - 90% of sulfate groups, a molecular weight of 5000 - 12000. The product has immunostimulating properties and shows antielastase activity. 5 C.p. f-crystals, 4 PL.

The invention concerns a method of obtaining a sulfated derivative galactan extracted from Klebsiella.

French patent OP N 2 574 429 describes azillion. extracted from Klebsiella, consisting of approximately 80% of neutral sugars, 20% lipid, less than 2% protein, with the GI - ical and immunomodulatory properties.

Application for French patent N 89.09305 of 11 July 1989 and the application for the European patent N 904019874 describe galactan low molecular weight extracted from Klebsiella, method of its production, its use as medicaments, in particular immunostimulatory drugs.

This invention relates to a method of obtaining a new sulfated derivative galactan extracted from Klebsiella from received above galactan.

The invention also aims at a method of obtaining a new sulfated derivative galactan extracted from Klebsiella, consisting mainly of neutral sulfated sugars, in the amount of 20-90% of hydrocision.

The composition of neutral sugars is determined by chromatography in the gaseous phase after methanolysis method ZANETTA J. Chromato (1972), 69, S. 291 or the way KAMERLING/Biochem J. (1975), 151, S. 491).

In particular, the invention aims at a method of obtaining a sulfated derivative galactan formed mainly of sulfated neutral sugars in the amount of 40-80% of the hydroxyl.

Among the products obtained by the method which is the subject of the invention, preferably sulfamidate 50-70% of the hydroxyl.

Sulfated derivative galactan obtained by the method of the invention, formed from a linear sequence of galactose 1-3 and has a molecular weight between 5000 and 12000 and preferably about 7,000.

These polysaccharides do not contain lipids and proteins.

The composition of galactose is determined by chromatography in the gaseous phase after recovery and methanolysis.

Lipids are determined by chromatography in the gaseous phase after methanolysis.

Proteins are determined by the method of LOWRY (J,B. C. (1951), 193, S. 265-273.

Link linear sequence galactose is determined by the conventional methods of methylation, chromatographic analysis in the gas phase, connected to a mass spectrometer, periodic oxidation, NMR from1H and13C. the Results show that the galactan formed from linear chains of galactose residues connected by the links of alpha (1-3) 50% and beta (1-3) 50% recurring element formed of 8 residues of galactopyranose and 1 residue galactofuranose.

Sulfated derivative galactan obtained by the method of the invention can be prepared from various species of Klebsiella. Especially interesting sulfated Priya Cultures of Microorganisms (CNCM) under NN 52145 and 1-163 and National Type Culture Collection under N 5055.

The method of the invention is that extracted from Klebsiella achillini, formed mainly of approximately 80% of neutral sugars, 20% lipid, less than 2% protein and has a molecular weight of about 12,500, treated with mild acid hydrolysis, chromatographic high yield on unionamerica, collect not retained fraction, is subjected to filtration on gel, collecting the fraction containing galactan mol.m. between 4000 and 10000, and sulfation thus obtained fraction galactan, purified by dialysis and produce thus obtained sulfated derivative galactan.

Extracted azillion from the original Klebsiella may be derived from bacterial water-soluble extract of Klebsiella by heating, and then the chromatographic fractionation. This receipt was already described in the French patent R 2 574 429 and in the German patent DE 3 543 267.

Bacterial water-soluble extract of Klebsiella was obtained by the method described in patent FR - 2 490 496 and EP . 49182.

In the preferred conditions of the invention:

- mild acidic hydrolysis azillion is conducted in a 1% solution of acetic acid is heated at 100about

then galactan is separated from the upper part of the chromatographic fractionation. Collect the fraction formed neutral sugars.

the fractionation can be performed on the chromatographic aminoalkenes, preferably of chromatogra - FIA with high output, for example on Magnum 9SAX Flipchart. Chromatography with high output unionamerica is that separate galactan by elution with water. The required fraction formed from the neutral sugars detected by spectrophotometric detection at 200 nm and 492 nm after staining break the phenol - sulfuric acid method of DUBOIS (Anal.Chem.(1956), 28, S. 350).

- this faction is made up of neutral sugars, is filtered on the gel, which allows you to collect the fraction containing galactan mol. m , located between 4000 and 10000. Filtering the gel is carried out in known carriers in the trade, such as Sephadex or Biogel of agarose; preferably use a Biogel A-1.5 M

- sulfation galactinol faction is using sulfonic acid, tkfont piperidine, acting by heating the reaction medium.

The dialysis is carried out with distilled water.

Sulfated derivative galactan obtained by the method which is the subject of this invention, has a very interesting pharmacological properties; in particular, it has excellent immunostimulating properties, and good tolerance. In particular, it has the property to bring education Il1and TNF (Tumor Necrosis Factor) at the level of the macrophage and the main thing is to stimulate the formation of free radicals (polycyclic). It also allows you to energyservice effect of GM-CSF (Granulo - monocyte - Colohy Stimulating Factor).

Mark is also a wonderful anti-Lactasoy activity against pancreatic elastase cattle, and against leukocyte elastase person. These products are also protivosvertyvayushchei activity.

These properties justify the use of sulfated derivative galactan, such as defined above, as medicines.

These drugs, for example, find application in the treatment or prevention of human diseases such as immunodepressed, infectious diseases caused by bacteria or virus is infectious, in the treatment follow-up and postoperative infections and allergies of all backgrounds. These medications can also be used in the treatment with bone marrow transplantation and spinal aplasia after treatment by chemical means.

The usual dose, changing depending on the use of the product, treat the patient and the disease may be, for example, from 0.5 to 5 mg per day by mouth.

These drugs are also used in pneumology at in the treatment of emphysema, lung inflammation, bronchitis, lung disorders caused by tabagismo, or atmospheric emissions in cardiology in the treatment of arthritis, as well as, for example, in dermatology for the treatment of psoriasis, burns, bolos and in aging skin in gastroenterology in the treatment of acute pancreatitis and in General in the treatment of all diseases associated with the operation of elastase, as well as in the treatment of thromboembolic diseases.

The usual dose, changing depending on the product, treat the patient and the disease may be, for example, 1-300 mg / day for a person intravenously.

Sulfated derivative galactan extracted from Klebsiella, such as defined above, can derived.

As medicines, sulfated derivative galactan extracted from Klebsiella, such as defined above, may be added to pharmaceutical compositions intended for the reception of the digestive tract, parenteral intake, or local way.

Corresponding pharmaceutical compositions can be, for example, solid or liquid, and is available in commonly used for a person's formulation, as for example, tablets, pills, solutions, syrups, suppositories, lyophilized or deliverydiovan drugs for injection, ovule, creams, ointments, liquids for external use, eye drops, aerosols; they are prepared in the usual way. One or more active principles can be put into the excipients usually used in these pharmaceutical compositions, such as talc, gum Arabic, lactose, starch, magnesium stearate, cocoa butter, non-aqueous or aqueous media, animal or vegetable fats, paraffin derivatives, glycols, various wetting, dispersancy, emulsifiers or preservatives.

P R I m e R 1. Sulfated derivative galactan extracted from Klebsiella. To pyridine, cooled to 0aboutWith ice, p, xtraining of Klebsiella, pneumoniae (25 mg) in suspension in pyridine (24 cm3). The mixture is heated 3 hours at 80aboutC, cooled to room temperature, diluted with distilled water (30 cm3and finally treated with 2.5 M sodium hydroxide solution (5 cm3). The resulting solution was concentrated in vacuo, deleteroute distiller - bath water for 3 days, frozen and lyophilizers.

Get sulfated derivative galactan extracted from Klebsiella.

The resulting product hydroxides sulfotyrosine in relation to 60 mol%.m. about 7,000.

Cooking galactan extracted from Klebsiella, pneumoniae. Dissolve 334 mg acylglycerol Klebsiella (obtained as in example 1 of French application No. 2 574 429 on the basis of strain inside the Institute Pasteur under N 1-163) in 33.4 cm31 - acetic acid solution and heated for 90 min at 100aboutC. After cooling, separating the precipitate containing lipidology fraction by centrifugation at 2000 G for 30 minutes Lyophilizer surface part and give you 200 mg of residue, which re-dissolved in 4 cm3water. Chromatographic factions in 1 cm3on the column for chromatography high-yield unionamerica Wat is the tion, containing neutral sugars found at 492 nm after sample phenol-sulfuric. Liofilizirovannam allocate 55 mg neutral faction.

The selection of galactan. Previous product lyophilization is dissolved in a buffer solution of water - acetic acid - pyridine (973 - 7 - 20), and then subjected to gel filtration chromatography on a column of Biogel A. 1.5 M (2 CM1.5 m) equilibrated with the same buffer solution. Collect the detected fraction of the breakdown of the phenol-sulphuric. Sterilized by filtration over a membrane of 0.22 μm, and then lyophilizer and obtain 22 mg of the target galactan.

P R I m m e R 2. Sulfated derivative galactan extracted from Klebsiella.

To pyridine, cooled in ice to 0aboutC, add 4 cm3chlorosulphonate. After returning to room temperature, add galactan extracted from Klebsiella, pneumoniae (obtained as described in preparation described in example 1) (85 mg) in suspension in pyridine (24 cm3). The mixture is heated for 2 hours at 80aboutC, cooled to room temperature and diluted with distilled water (30 cm3). Bring the pH of the reaction mixture to 6 by the addition of 2.5 M sodium hydroxide solution. The resulting solution is subjected to di is one derived galactan, extracted from Klebsiella, pneumoniae.

Biological research

The activity of the sulfated derivative galactan extracted from Klebsiella, determined by measuring the inhibition of sincity Gruters and cells. Nature 330 Tom S. 74-77, 5 November 1987) and the definition of the protecting activity shown against some cell cultures.

(A) Preparation and titration of the virus.

1) Preparation of the virus.

the floating part of the cells N 9 111in(chronically infected H1V) diluted within 48 hours from 105cells/cm3.

- spin-on floating part, filtered at 0.45 mcmole containing residue and stored at -80aboutC.

2) Titration of the virus.

- reverse transcriptase inhibitors (RT)

Fresh floating part 4,0106pulse/min/cm3< / BR>
After thawing 4,5106pulse/min/cm3< / BR>
- experience MT4/MTT

Technique:

100 μl of a dilution series of virus in the disc, micrometrology

- the addition of 100 μl of cell suspension containing 5104cells MT4; -

culture 7 days at 37aboutC, 5% CO2;

- increase MTT (salt tetrazolium dye viability)

the reference optical density (DO) at 540 nm (after solution of the crystals).

Results. Determine the ratio of the optical density of infected cells (cytotoxicity virus = weak D) to the optical density of infected cells (viability and D maximum) depending on the dilution of the virus (the average number of 3 ways).

The more cells are infected and the weaker the relationship.

B) Protective activity of sulfated derivative galactan extracted from Klebsiella.

1) Braking Sincity.

Produce a joint cell culture N 9 111 (chronically infected 105cells (cm3) cells SUP T1 (not infected: 2105cells/cm3).

Formed of syncytia that counts in the microscope.

Each type of cells preincubated or neprincipijelan with the studied product one night at 37aboutC.

If there is no preincubation, the target product is added to the cells, when produce culture.

When there is inhibition of the formation of Syncytia in the treated cells relative to control cells, then the studied product is a defender.

2) Experience MT/MTT

Incubated at 37aboutWith 7 days, add MTT and counting on the spectrophotometer.

Determine the percent inhibition of infection of MT4 cells.

The results obtained are given in table. 3.

3) Experience N 9.

Use of H9 cells that process polybrene, washed, picked up in 100 μl of medium with or without the product studied various concentrations.

Incubated for 2 h at 37aboutC, add 100 μl of the HIV virus at 1/10, incubated 1 h at 37aboutWith washed.

Cells take in 1 cm3environment with or without the product studied, distributed on 2 way 500 ál of the path and 2105cells on the path.

In this day + 4 add 500 ál of feed medium with or without the studied product.

In this day + 8 replace half of the floating part of the new environment.

In this day +11 produce the dosage of reverse transcriptase inhibitors on the floating part and defining immunofluorescence assay on the cells.

The results obtained in various experiments show that the studied product significantly inhibits Albania protivoglistotnoe activity.

Protivolakozna activity was determined by spectrophotometric dosage against leukocyte elastase person (a technique similar to that described Boudier and co. Clin. Chim. Acta. 132 - 309-315 (1985), see below Product Human leukocyte example

elastase 1 Ki : 110-8M

1. The WAY to OBTAIN SULFATED DERIVATIVE GALACTAN, namely, that of azillion extracted from bacteria Klebsiella pneumoniae (mol.m. about 12,500 and formed from approximately 80% neutral carbohydrate, 20% lipids, and 2% protein, subjected to mild acid hydrolysis, chromatographic on aminoalkenes, lyophilizer carbohydrate fraction, subjecting it gel filtration, collecting the fraction containing galactan mol.m. 4000 - 10000, lyophilizers, conduct sulfation of the resulting fractions galactan, then the reaction mixture was subjected to dialysis and obtain the target product with the following characteristics: formed by the linear sequence of residues of galactose connected by the links (1 - 3) (methods of analysis: methylation, controllable periodic destruction oxidation spectrometry); mol.m. 5000 - 12000; contains 20 to 90% of sulfate groups.

2. The method according to p. 1, characterized in that the sulfation faction ha is P>C.

3. The method according to p. 1, characterized in that the dialysis of the reaction mixture are against distilled water.

4. The method according to p. 1, characterized in that the target product contains 40 to 80% of the sulfate groups.

5. The method according to p. 1, characterized in that the target product contains 50 - 70% of sulfate groups.

6. The method according to p. 1, characterized in that as the initial use of azillion extracted from bacterial strains of Klebsiella pneumoniae, located at the Institut Pasteur in Paris (or in the National Collection of Cultures of microorganisms) under NN 52145 and 1-163 and the National collection of type culture under N 5055.

 

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