GB2060645A

GB2060645A - Glycoproteins extracted from Klebsiella pneumoniae

Info

Publication number: GB2060645A
Application number: GB8024949A
Authority: GB
Original assignee: Roussel Uclaf SA
Current assignee: Sanofi Aventis France
Priority date: 1979-07-31
Filing date: 1980-07-30
Publication date: 1981-05-07
Also published as: GB2060645B; CH645130A5; FR2462477B1; DE3029111A1; JPS6363560B2; NL8004406A; FR2462477A1; JPS5622793A; DE3029111C2

Abstract

Water-soluble glycoproteins extracted from Klebsiella pneumoniae, characterised in that they contain from 10% to 20% of proteins, 50% to 70% of neutral oses, 15% to 25% of glucuronic acid and 1% to 2% of osamines and have a molecular weight of from 80,000 to 350,000 daltons, processes for obtaining them and pharmaceutical compositions containing them are described. The glycoproteins have anti-bacterial and immunostimulant properties.

Description

SPECIFICATION Glycoproteins extracted from Klebsiella pneumoniae The present invention relates to new glycoproteins extracted form Klebsiella pneumoniae, to a process for obtaining them, to their use as medicaments and to compositions containing them.

Certain glycoproteins extracted from Klebsiella pneumoniae have been previously described in, for example, French Patents Nos. 2,043,475,2,088,112 and 2,171,907.

The present Application relates to more precisely defined glycoproteins. Thus according to one feature of the present invention there are provided water-soluble glycoproteins extracted from Klebsiella pneumoniae, characterised in that they contain from 10% to 20% of proteins, 50% to 70% of neutral oses, 15% to 25% of giucuronic acid and 1% to 2% of osamines and have a molecular weight of from 80,000 to 350,000 daltons.

Neutral oses include, for example, in particular neutral hexoses such as glucose, mannose and galactose.

Preferred glycoproteins according to the invention are those, having a molecular weight, estimated by ultracentrifuging, of about 100,000 daltons.

The glycoproteins of the invention can be extracted from different strains of Klebsiella pneumoniae; however, preferred glycoproteins are those extracted from the strain deposited at the Institut PASTEUR under the number 52,145.

A study of the structure of these glycoproteins using different chemical techniques, especially reduction of uronic acid residues with carbodiimide, permethylation, uronic degradation, periodic oxidation and oxidation with chromic oxide, have enabled the composition and the structure of the products of the present Application to be specified.

The glycoproteins according to the invention consist of a protein chain on to which is grafted the polysaccharide fraction.

The preferred glycoproteins according to the invention are those for which the protein fraction is composed of about 30% of acidic amino acids. By acidic amino acids is meant amino acids possessing a side chain containing a C00H group, such as aspartic acid or glutamic acid.

Preferred glycoproteins according to the invention include those in which the N-terminal amino acid of the protein fraction is aspartic acid.

Also preferred are glycoproteins according to the invention in 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 per one molecule of galactose.

Among the latter, particularly preferred glycoproteins are those for which the polysaccharide fraction is essentially composed of the repetition of a tetrasaccharide unit of which the structure is as follows:

Among the new glycoproteins which form the subject of the invention, particular mention should be made of those in which the polysaccharide fraction is formed of two polysaccharide chains, each of which is bonded to the protein fraction by an N-glycoside bond between a molecule of glucosamine at one end of the polysaccharide chain and a molecule of asparagine of the protein chain.

According to a further feature of the present invention there is provided a process for obtaining glycoproteins according to the invention as hereinbefore defined characterised in that a solution of glycoproteins obtained from an extract of cultures of Klebsiella pneumoniae is treated with a quaternary ammonium, the precipitate obtained is isolated then dissolved in an aqueous solution of sodium chloride, the saline solution of glycoproteins thus obtained is treated cold with an alkanol of low molecular weight and a new precipitate is thus obtained which may, if desired, then be re-dissolved in water, dialysed and lyophilised.

Different solutions of glycoproteins can be used as starting materials, and these solutions may for example be obtained by diafiltration of lysates from cultures of Klebsiella pneumoniae on calibrated porous membranes capable of retaining molecules of a molecular weight equal to or greater than a given molecular weight, which is a constant for these membranes.

The membranes used can, for example, be the membranes sold under the brand names AMICON XM50, PM30 and UM2, but it is preferred to use membranes for which the retention theshold is 100,000 daltons, such as the membranes sold under the name XM 100 or H 1 P 100, by the company AM ICON. The particularly-preferred membranes enable molecules to be retained of which the molecular weight is greater than 300,000 daltons; these are advantageously constituted by the membranes sold under the name XM300 by the companies AM ICON and ROMICON.

Diafiltration enables molecules of which the molecular weight is greater than a given molecular weight to be selected in solution by the choice of membrane, depending upon the retention threshold desired. It will be apparent to those skilled in the art that the use of other solutions having the same characteristics, obtained by other means, such as, for example, by chromatography on hydrophilic polymerised gel, is quite possible.

Previously to this selection operation the lysate can, very advantageously, be freed from lipids and its nucleic acids.

Under particularly-preferred conditions for carrying out the process according to the invention the starting solution of glycoproteins is obtained according to the process described in the second Certificate of Addition No. 2,171,907 to the French Patent No. 2,043,475. In this Certificate of Addition the molecular weight of the glycoproteins has been estimated by the technique of exclusion of gels.

The quaternary ammonium which is used to treat the starting solution of glycoproteins can be, for example, cetylpyridylammonium chloride, but the use of cetyltrimethylammonium bromide or Cetavlon is particularly preferred.

After treatment with quaternary ammonium the precipitate obtained can be separated from the supernatant liquid by standard procedures such as decanting or filtering, but it is preferred to separate it by centrifuging.

The precipitate is then advantageously re-dissolved using a 0.2M solution of sodium chloride.

The solution obtained is then treated, cold, at about +40C, using an alkanol of low molecular weight such as methanol, ethanol, n-propanol or isopropanol, but preferably ethanol is used. The most interesting results are obtained by using six volumes of ethanol to one volume of saline solution, for one night, at the temperature of +40 C.

The precipitate can then be re-dissolved in water and preferably dialysed to purify it. The dialysis is carried out using cells of standard type closed by membranes, for example of collodion or of cellulose.

Preferred cells are those of which the membrane is of regenerated cellulose, with an average pore diameter equal to about 240A, retaining substances of which the molecular weight is greater than a value ranging from 12 to 14,000 daltons. Suitable dialysis cells include, for example, VISKING tubes.

Dialysis enables impurities of low molecular weight remaining, e.g. traces of alkanol, to be removed from the solution of glycoproteins.

The glycoproteins of the invention can, however, be obtained in slightly less pure form without carrying out the dialysis stage.

In a preferred embodiment of the process according to the present invention, the quaternary ammonium used is cetyltrimethylammonium bromide; the first precipitate is isolated by centrifuging; and the alkanol of low molecular weight is ethanol.

Glycoproteins obtained by a process according to the present invention as hereinbefore defined constitute a further feature of the present invention.

The products of the present invention possess very interesting pharmacological properties; they are endowed, especially, with remarkable anti-bacterial and immunostimulant properties as well as very good tolerance.

These properties are illustrated hereinafter in the experimental portion.

The glycoproteins of the present invention may accordingly be used as medicaments; glycoproteins according to the invention as hereinbefore defined for use as medicaments constitute a further feature of the invention.

The glyceproteins according to the invention can, for example, be used in the treatment or the prevention, in man and in animals, of infectious diseases caused by bacteria or viruses, in the treatment of diseases caused by parasites and of toxi-infections and in the treatment of post-hospitalisation and post-surgical infections.

The usual dose, which can be varied according to the product used, the subject treated and the complaint concerned, can be, for example, in man from 0.5 mg to 10 mg per day by oral route, from 2 to 10 mg per day by rectal route and from 0.25 to 5 mg per day by parenteral route.

According to yet a further feature of the present invention there are provided pharmaceutical compositions containing as active ingredient at least one glycoprotein according to the invention as hereinbefore defined.

Compositions according to the invention can be administered by digestive, parenteral or local routes.

The compositions according to the present invention may, for example, be solid or liquid and can be presented in the pharmaceutical forms currently used in human medicine such as, for example, plain or sugar-coated compressed tablets, gelatin capsules, granules, solutions, syrups, suppositories, injectable preparations, ovules, creams, ointments, lotions, drops and collyria; such compositions can be prepared according to conventional methods. The active ingredient(s) can be mixed with excipients usually employed in these pharmaceutical compositions such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting, dispersing or emulsifying agents and/or preservatives. The compositions according to the present invention can conveniently be presented in the form of dosage units, each dosage unit containing, for example, from 0.25 to 5 mg of active ingredient.

The following non-limiting examples iilustrate the present invention: EXAMPLE 1 20 g of product is obtained in Example 1 of French Patent No. 2,171,907 are dissolved in two litres of softened water.

About 1.65 1 of 3% Cetavlon is added slowly and the mixture is agitated for one hour then centrifuged at 10,000 rpm for 1 5 minutes.

The precipitate is taken up with 0.5 1 of a 0.2M solution of sodium chloride. 3 1 of 950 ethanol is then added over 1 5 minutes. After one hour's agitation, the mixture is centrifuged at 10,000 rpm for 1 5 minutes, the supernatant liquid thus obtained is removed and the precipitate is re-dissolved in 1 litre of water then dialysed for 48 hours in Visking tubes against softened water at +40C. At the erid of this dialysis the solution is lyophilised. 8.16 g of the desired glycoproteins are obtained.

EXAMPLE 2 20 g of product as obtained in Example 1 of French Patent No. 2,171,907 are dissolved in 1 litre of softened water. 0.800 litres of 3% Cetavlon are added with agitation. After one hour's agitation, the mixture is centrifuged at 10,000 rpm for 1 5 minutes.

The precipitate thus obtained is re-dissolved in 0.250 litre of a 0.2M solution of sodium chloride.

1.5 litres of 950 ethanol is added with agitation. After one hour's agitation the mixture is centrifuged for 1 5 minutes at 10,000 rpm. The supernatant liquid is removed and the precipitate taken up in 0.500 litre of water and dialysed for 48 hours in Visking tubes against softened water at +40C.

At the end of this dialysis, the solution is lyophilised and 9.4 g of the desired giycoproteins sought are obtained.

EXAMPLE 3 Compressed tablets having the fol!owing formulation are prepared: glycoproteins obtained in Example 1 5 mg excipient q.s. for one compressed tablet up to 100 mg (Detail of the excipient: lactose, starch, talc, magnesium stearate).

EXAMPLE 4 An ointment having the following formulation is prepared: glycoproteins obtained in Example 1 200 mg excipient q.s. for 100 g Pharmacological study A Antibacterial activity of the glycoproteins obtained according to the method of carrying out the present application The glycoproteins of the invention bring about intense and lasting antibacterial protection at very small doses. This activity is polyvalent and is exerted both against Gram positive germs and against Gram negative germs. The activity is more preventative than curative.

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

At a dose of 1 Oy/kg the "glycoproteins" of Examples 1 and 2 ensure 100% protection against an infection corresponding to 500 LD 50 of Klebsiella pneumoniae. At the same dose and for the same products the protection against an infection corresponding to 12,500 LD 50 of Klebsiella pneumoniae is 85%. At a dose of 1 OOy/kg protection against 12,500 LD 50 of this same micro-organism is 100%.

The antibacterial activity with respect to Klebsiella pneumoniae varies depending upon the moment of administration of the glycoproteins: according to whether they are administered forty-eight or ninety-six hours before the infection the percentages of protection are respectively 30% and 100%; the glycoproteins according to the invention possess, therefore, very ciear antibacterial activity as a preventative.

B Stimulation of the non-specific defences This stimulation was studied using the test of "clearance" with carbon in the mouse, being inspired by the technique perfected by HALPERN, which consists of injecting into the ocular sinus of the animal a suspension of colloidal carbon and in evaluating as a function of time the kinetics of the disappearance of the carbon in the blood, by carrying out measurements of optical density.

The products are administered to the animal by intraperitoneal route, twenty-four and forty-eight hours before the test and the results are expressed in percentage activity with reference to controls which have received, solely, an injection of colloidal carbon.

Controls % activity 30 minutes Product 8 minutes after the of Example Doses after the injection injection 1 0.25 mg/kg 42% 65% 2 0.25 mg/kg - 43% 65% Examination of these results enables the intense stimulation; caused by these two products, of the defences of the organism to be noted.

C Acute toxicity The lethal dose 50 (LD 50) by intraperitoneal route in the mouse was determined by the method of BEHRENS and KARBER.

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

D Tolerance The sub-cutaneous injection of 0.2 ml of glycoproteins of Examples 1 and 2, at the dose of 1 ,000y/kg, in the mouse, does nct cause any local or general intolerance.

Claims

1. Water-soluble glycoproteins extracted from Klebsiella pneumoniae, characterised in that-they contain from 10% to 20% of proteins, 50% to 70% of neutral oses, 1 5% to 25% of glucuronic acid and 1% to 2% of osamines and have a molecular weight of from 80,000 to 350,000 daltons.

2. Glycoproteins as claimed in claim 1 having a molecular weight of about 100,000 daltons.

3. Glycoproteins as claimed in claim 1 or claim 2 characterised in that they are extracted from the strain of Klebsiella pneumoniae deposited at the Institut PASTEUR under number 52,145.

4. Glycoproteins as claimed in any one of the preceding claims wherein the protein fraction contains about 30% of acidic amino acids as defined herein.

5. Glycoproteins as claimed in any one of the preceding claims wherein the N-terminal amino acid of the protein fraction is aspartic acid.

6. Glycoproteins as claimed in any one of the preceding claims wherein 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 per one molecule of galactose.

7. Glycoproteins as claimed in claim 6 wherein the polysaccharide fraction consists essentially of a tetrasaccharide repeating unit of which the structure is:

glucose 1 4mannose 1 4glucose 1 3 1 glucuronic acid

8. Glycoproteins as claimed in any one of the preceding claims wherein the polysaccharide fraction is formed of two polysaccharide chains each of which is bonded to the protein fraction by an Nglucoside bond between a molecule of glucosamine at one end of the polysaccharide chain and a molecule of asparagine of the protein chain.

9. Glycoproteins as claimed in claim 1 substantially as herein described.

10. A process for obtaining glycoproteins as claimed in any one of the preceding claims, characterised in that a solution of glycoproteins obtained from an extract of cultures of Klebsiella pneumoniae is treated with a quaternary ammonium, the precipitate obtained is isolated then dissolved in an aqueous solution of sodium chloride, the saline solution of glycoproteins thus obtained is treated cold with an alkanol of low molecular weight and a new precipitate is thus obtained.

11. A process as claimed in claim 10 wherein the starting solution of glycoproteins is obtained by diafiltration of a lysate extract of cultures of Klebsiella pneumoniae.

1 2. A process as claimed in claim 11 wherein the lysate is first freed from lipids and nucieic acids.

13. A process as claimed in any one of claims 10 to 12 wherein the precipitate obtained on treatment with alkanol is subsequently re-dissolved in water, dialysed and lyophilised.

14. A process as claimed in any one of claims 10 to 13 wherein the quaternary ammonium is cetyltrimethylammonium bromide.

1 5. A process as claimed in any one of claims 10 to 14 wherein the first precipitate is isolated by centrifugation.

1 6. A process as claimed in any one of claims 10 to 1 5 wherein the alkanol of low molecular weight is ethanol.

17. A process as claimed in claim 1 6 wherein six volumes of ethanol to one of saline solution are used.

18. A process as claimed in any one of claims 10 to 1 7 wherein the first precipitate is dissolved in a 0.2M solution of sodium chloride.

19. A process for obtaining glycoproteins as claimed in clairn 1 substantially as herein defined in either of Examples 1 and 2.

20. Glycoproteins whenever obtained by a process as claimed in any of claims 10 to 1 9.

21. Pharmaceutical compositions containing as active ingredient at least one glycoprotein as claimed in any one of claims 1 to 9 and 20.

22. Compositions as claimed in claim 21 in a form suitable for digestive, parenteral or local administration.

23. Compositions as claimed in claim 21 or claim 22 in the form of dosage units.

24. Compositions as claimed in claim 23 wherein each dosage unit contains from 0.25 to 5 mg of active ingredient.

25. Pharmaceutical compositions substantially as herein described in either of Examples 3 and 4.

26. Glycoproteins as claimed in any one of claims 1 to 9 and 20 for use as medicaments.

27. Glycoproteins as claimed in any one of claims 1 to 9 and 20 for use in the treatment or prevention of infectious diseases or in the treatment of diseases caused by parasites, toxi-infections or post-hospitalisation or post-surgical infections.

28. Each and every product, process, method and composition herein disclosed.