CH645130A5 - GLYCOPROTEINS OF KLEBSIELLA PNEUMONIAE, PROCESS FOR OBTAINING SAME AND MEDICINAL PRODUCTS CONTAINING THEM. - Google Patents

Description

The present invention relates to new glycoproteins extracted from Klebsiella Pneumoniae, the process for obtaining them and the medicaments containing them.

A number of French patents have already described glycoproteins extracted from Klebsiella pneumoniae, this is the case, for example, French patents 2,043,475, 2,088,112, and 2,171,907.

The present invention relates to more precisely defined glycoproteins and thus relates to new water-soluble glycoproteins extracted from Klebsiella Pneumoniae, characterized in that they contain 10% to 20% of proteins, 50% to 70% of neutral dares, 15 % to 25% glucuronic acid, 1% to 2% osamines and have a molecular weight between 80,000 and 350,000 daltons.

Neutral dares are used in particular to denote neutral hexoses such as glucose, mannose or galactose.

The glycoproteins as defined above are preferably used, characterized in that their molecular weight estimated by ultra centrifugation is approximately 100,000 daltons.

The glycoproteins of the invention can be extracted from different strains of Klebsiella pneumoniae; however, we particularly retain those which come from the already known strain deposited at the PASTEUR Institute under the number 52 145.

The study of the structure of these glycoproteins using different chemical techniques, in particular, the reduction of uronic acid residues by carbodiimide, permethy-lation, uronic degradation, periodic oxidation or oxidation by chromium oxide, made it possible to specify the composition and structure of the products of the present invention.

The glycoproteins according to the invention are formed from a protein chain onto which the polysaccharide fraction is grafted.

The preferred glycoproteins according to the invention are those for which the protein fraction is composed of approximately 30% of acidic amino acids. The term “amino acid acids” is understood to mean amino acids such as aspartic acid or glutamic acid, that is to say amino acids having a side chain comprising a COOH group.

The glycoproteins according to the invention are also preferably retained for which the N-terminal amino acid of the protein fraction is aspartic acid.

The preferred glycoproteins according to the invention are also those potar which the polysaccharide fraction contains approximately from 9.5 to 10.5 molecules of glucose, from 4 to 5 molecules of mannose and from 3 to 3.5 molecules of glucuronic acid for a galactose molecule.

Among these, we particularly note those for which the polysaccharide fraction is essentially composed of the repetition of the tetrasaccharide unit, the structure of which is as follows:

—F-5-gluöose- - mannose - glucose *]

i 3

I 1

glucuronic acid

Among the new glycoproteins which are the subject of the invention, there are also very particularly those whose frac-

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polysaccharide is made up of two polysaccharide chains, each of which is linked to the protein fraction by an N-glycosidic link between a glucos-amine molecule at one end of the polysaccharide chain and an asparagine molecule in the protein chain.

The invention also relates to a process for obtaining new water-soluble glycoproteins as defined above; said process is characterized in that a solution of glycoproteins obtained by diafiltration of a lysate extract from cultures of Klebsiella pneumoniae is treated with quaternary ammonium, isolates and then dissolves the precipitate obtained, with using an aqueous sodium chloride solution, cold treatment of the saline glycoprotein solution thus obtained using a low molecular weight alkanol, thus obtains a new precipitate which is redissolved in water, dialysis then freeze-dried.

Different glycoprotein solutions can be used at the start of the process, these solutions are obtained by diafiltration of lysates from Klebsiella pneumoniae cultures on calibrated porous membranes, capable of retaining molecules with a molecular weight equal to or greater than a given molecular weight, which is a constant for these membranes.

The membranes used are, for example, the membranes sold under the brand names AMICON XM50, PM30 and UM2, but it is preferred to use membranes with a retention threshold of 100,000 daltons, such as the membranes sold under the designation XM 100. or Hl PI00, by the company AMICON. The very particularly preferred membranes make it possible to retain molecules whose molecular weight is greater than 300,000 daltons; these are advantageously constituted by the membranes sold under the designation XM300 by the companies AMICON and ROMICON.

Diafiltration makes it possible to select, in solution, molecules whose molecular weight is greater than a given molecular weight, as has been said, by the choice of the membrane, as a function of the desired retention threshold. It is obvious to those skilled in the art that the use of other solutions of the same characteristics, obtained by other means, such as for example by chromatography on hydrophilic polymerized gel, is entirely possible.

Prior to this selection operation, the lysate can, very advantageously, be defatted and freed of its nucleic acids.

In very particularly preferred conditions for carrying out the process according to the invention, the starting glycoprotein solution is obtained according to the process described in the second certificate of addition no. 2,171,907 to French patent no. 2,043,475; in this addition certificate, the molecular weight of the glycoproteins was estimated by the gel exclusion technique.

The quaternary ammonium which is used to treat the starting glycoprotein solution can be, for example, pyridyl-cetyl-ammonium chloride, but very particularly, trimethyl cetyl ammonium bromide or Ketavlon.

After treatment with quaternary ammonium, the precipitate obtained can be separated from the supernatant by conventional methods, such as decantation or filtration, but it is preferred to separate it by centrifugation.

The precipitate is then advantageously redissolved using a 0.2 M sodium chloride solution.

The solution obtained is then treated cold, at about + 4 ° C., using a low molecular weight alkanol such as methanol, ethanol, n-propanol or isopropanol, but use is made preferably ethanol. The most interesting results are obtained by using six volumes of ethanol for one volume of saline overnight, at a temperature of + 4 ° C.

The precipitate is redissolved in water and dialyzed to purify it. Dialysis is carried out using cells of the conventional type closed with membranes, for example, collodion or cellulose. We retain, in particular, cells whose membrane is made of regenerated cellulose, with an average pore diameter equal to about 24Å, retaining substances whose molecular weight is greater than a value ranging from 12 to 14,000 daltons. Among the dialysis cells, we can particularly note the VISKING tubes.

io This dialysis makes it possible to remove from the glycoprotein solution, the remaining low molecular weight impurities, such as traces of alkanol.

Obviously, the slightly less pure glycoproteins of the invention can be obtained by not carrying out the dialysis step.

Under these preferred implementation conditions, the process described above is characterized in that:

- the quaternary ammonium used is cetyl-trimethyl-ammonium bromide;

- the first precipitate is isolated by centrifugation;

- the low molecular weight alkanol is ethanol.

The present invention also relates to the glycoproteins as obtained by the process according to the invention.

The products which are the subject of the present invention have very advantageous pharmacological properties; they are endowed, in particular, with remarkable antibacterial and immunostimulatory properties, as well as very good tolerance.

These properties are illustrated later in the experimental part.

These properties justify the use of the glycoproteins of the present invention, as medicaments. The present invention thus also relates to the use, as medicaments, of glycoproteins as defined above. Among the medicaments of the invention, there are also those characterized in that they consist of the glycoproteins obtained by the method of the invention.

These drugs find, for example, their use in the treatment or prevention, in humans and animals, of 40 infectious diseases caused by bacteria or viruses, in the treatment of parasitic diseases, toxi-infections. tions, in the treatment of post-hospital and post-surgical infections:

The usual dose, which varies according to the product used, the subject treated and the condition in question, can be, for example in humans from 0.5 mg to 10 mg per day, orally, from 2 to 10 mg 0.25 to 5 mg per day, rectally, parenterally.

The invention finally relates to the pharmaceutical compositions which contain the glycoproteins of the present invention, as active ingredient.

As medicaments, the glycoproteins of the present invention can be administered by the digestive, parenteral or local routes.

The corresponding pharmaceutical compositions can be, for example, solid or liquid and can be in the pharmaceutical forms commonly used in human medicine, such as, for example, tablets, simple or coated, capsules, granules, solutions, syrups, suppositories, injections, ova, creams, ointments, lotions, drops, eye drops; they are prepared according to the usual methods. The active ingredient (s) can be incorporated therein into excipients commonly used in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, vehicles aqueous or not, fatty substances of animal or vegetable origin, derivatives

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paraffinic, glycols, various wetting, dispersing or emulsifying agents, preservatives.

It will now be given, without limitation, examples of implementation of the invention.

Example 1

20 g of product as obtained in Example No. 1 of French Patent No. 2,171,907, are dissolved in two liters of deionized water.

About 1.61 of 3% Cetavlon is added slowly, the whole is stirred for one hour, then centrifuged at 10,000 rpm for 15 minutes.

The precipitate is taken up in 0.51 of a 0.2 M solution of sodium chloride. Then 31 of 95 ° ethanol is added over 15 minutes. After one hour of stirring, centrifuged at 10,000 rpm for 15 minutes, the supernatant thus obtained is removed and the precipitate is redissolved in 11 water then dialyzed for 48 hours in Visking tubes against permuted water at + 4 ° C. At the end of this dialysis, the solution is lyophilized. 8.16 g of the sought-after glycoproteins are obtained.

Example 2

20 g of product as obtained in Example 1 of French Patent No. 2,171,907, are dissolved in 11 of deionized water. 0.8001 of 3% Ketavlon is added with stirring. After one hour of stirring, centrifugation at 10,000 rpm, for 15 minutes.

The precipitate thus obtained is redissolved in 0.2501 of a 0.2 M solution of sodium chloride. With stirring, 1.51 of 95 ° ethanol are added. After one hour of stirring, centrifugation is carried out for 15 minutes at 10,000 rpm. The supernatant is removed and the precipitate taken up in 0.5001 of water and dialyzed for 48 hours in Visking tubes against water deionized at + 4 ° C.

At the end of this dialysis, the solution is lyophilized and 9.4 g of the glycoproteins sought are obtained.

Example 3

Tablets corresponding to the formula have been prepared:

- glycoproteins obtained in Example 1 5 mg 40 1

- excipient q.s. for a tablet finished at 100 mg 2 (Details of excipient: lactose, starch, talc, magnesium stearate).

Example 4

We prepared an ointment with the formula:

- glycoproteins obtained in Example 2 200 mg

- excipient q.s.p. 100 g my Gram + than on Gram - germs. The action is more preventive than curative.

The product is administered to mice before the injection of the germs.

5 At a dose of 10 y / kg, the "glycoproteins" of Examples 1 and 2 provide a percentage of protection of 100% against an infection corresponding to 500 LD 50 of Klebsiella pneumoniae. At the same dose and for the same products, the protection is 85% against an infection corresponding to io 12 500 LD 50 of Klebsiella pneumoniae. At a dose of 100 y / kg it is 100% against 12,500 LD 50 of this same microorganism.

The antibacterial activity with respect to Klebsiella pneumo-i5 niae varies depending on the time of administration of the glycoproteins, depending on whether they are administered forty-eight or ninety-six hours before infection, the percentages of protection are respectively 30% and 100%; the glycoproteins according to the invention therefore have a very clear anti-bacterial action as a preventive measure.

B. Stimulation of non-specific defenses.

This stimulation was studied on the carbon “clearance” test in mice, inspired by the technique developed by HALPERN, which consists in injecting into the ocular sinus a suspension of colloidal carbon in the animal and to assess, as a function of time, the kinetics of the disappearance of carbon in the blood, by carrying out optical density measurements.

n / a The products are administered to the animal intraperitoneally, twenty-four and forty-eight hours before the test and the results are expressed as a percentage of activity compared to controls which have received, only, the injection of colloidal carbon.

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PRODUCT DOSES% of activity Witnesses of 8 minutes 30 minutes the example: after the injection after the injection.

0.25 mg / kg 0.25 mg / kg

42% 43%

65% 65%

Pharmacological study

A. Antibacterial activity of the glycoproteins obtained according to the mode of execution of the present application.

The glycoproteins of the invention trigger intense and lasting antibacterial protection at very low doses. This action is versatile and works on both managers and

Examination of these results allows us to note the intense stimulation caused by these two products, on the body's defenses 45.

C. Acute toxicity

The lethal dose 50 (LD 50) intraperitoneally, in mice, was determined by the method of BEHRENS and so KÄRBER.

It is 100 mg / kg for the glycoproteins of Examples 1 and 2.

D. Tolerance

The subcutaneous injection of 0.2 ml of glycoproteins from ss examples 1 and 2, at a dose of 1000 y / kg, in mice, does not cause any local or general intolerance.

VS

Claims (16)

645130 2 1. Water-soluble glycoproteins extracted from Klebsiella pneumoniae, characterized in that they contain 10% to 20% of proteins, 50% to 70% of neutral dares, 15% to 25% of glucuronic acid, 1% to 2% of osamines and have a molecular weight between 80,000 and 350,000 daltons. 2. Glycoproteins according to claim 1, characterized in that their molecular weight is approximately 100,000 daltons. 3. Glycoproteins according to claim 1 or 2, characterized in that they are extracted from the strain deposited at the PASTEUR Institute, under the number 52 145. 4. Glycoproteins according to one of claims 1 to 3, characterized in that the protein fraction is composed of about 30% acid amino acids. 5. Glycoproteins according to one of claims 1 to 4, characterized in that the N-terminal amino acid of the protein fraction is aspartic acid. 6. Glycoproteins according to one of claims 1 to 5, characterized in that the polysaccharide fraction contains from 9.5 to 10.5 molecules of glucose, from 4 to 5 molecules of man-nose and from 3 to 3.5 molecules glucuronic acid, for a galactose molecule. 7. Glycoproteins, according to claim 6, characterized in that the polysaccharide fraction is essentially composed of the repetition of the tetrasaccharide unit, the structure of which is as follows: [3 glucose i - mannose -glucose - ^) - 13 11 glucuronic acid 8. Glycoproteins, according to one of claims 1 to 7, characterized in that the polysaccharide fraction is formed of two polysaccharide chains each of which is linked to the protein fraction by an N-glycosidic bond between a molecule of glucosamine d an end of the polysaccharide chain and an asparagine molecule of the protein chain. 9. Process for obtaining the glycoproteins defined in one of claims 1 to 8, characterized in that a solution of glycoproteins obtained by diafiltration of an extract of culture lysate of Klebsiella pneumoniae, isolates then dissolves the precipitate obtained, using an aqueous solution of sodium chloride, cold treats the saline solution of glycoproteins thus obtained, using a light alkanol molecular, thus obtains a new precipitate which is redissolved in water, dialysis then lyophilized. 10. Method according to claim 9, characterized in that - the quaternary ammonium used is cetyl-trimethyl-ammonium bromide; - the first precipitate is isolated by centrifugation; - the low molecular weight alkanol is ethanol. 11. The new glycoproteins obtained by the process according to claim 9 or 10. 12. Medicament constituted, as active principle, by the water-soluble glycoproteins according to claim 1 or 2. 13. The medicament according to claim 12, consisting of the water-soluble glycoproteins defined in one of claims 3 to 8. 14. The medicament according to claim 12, consisting of the water-soluble glycoproteins defined in claim 11. 15. The medicament according to claim 12 or 13, in the form of a pharmaceutical composition. 16. Medicament according to claim 14, in the form of a pharmaceutical composition.