IE56612B1 - New process for the preparation of immuno-stimulating acylglycoproteins extracted from klebsiella pneumoniae and anti-allergic pharmaceutical compositions - Google Patents

Abstract

For the Contracting States BE CH DE GB IT LI LU NL SE 1. Preparation process for glycoproteins in which, in order, a solution of purified glycoproteins is prepared by diafiltration of an extract of a lysate of cultures of Klebsiella pneumoniae, the said solution is treated with a halide of a quaternary ammonium, the supernatant is isolated by eliminating the precipitate so obtined, the supernatant is concentrated by use of at least one means of selection of molecules, the concentrate is treated cold with at least one alkanol of low molecular weight, the precipitate so obtained is isolated and, if desired, is washed with at least one alkanol of low molecular weight and dried. For the Contracting State AT 1. Preparation process for glycoproteins in which, in order, a solution of purified glycoproteins is prepared by dialfiltration of an extract of a lysate of cultures of Klebsiella pneumoniae, the said solution is treated with a halide of a quaternary ammonium, the supernatant is isolated by eliminating the precipitate so obtained, the supernatant is concentrated by use of at least one means of selection of molelules, the concentrate is treated cold with at least one alkanol of low molecular weight, the precipitate so obtained is isolated and, if desired, is washed with at least one alkanol of low molecular weight and dried.

Description

The present invention relates to a process for the preparation of immune-stimulating acylglycoproteins extracted from Klebsiella pneumoniae as well as to anti-allergic pharmaceutical compositions containing acylglycoproteins prepared by such a process.

French Patent Application No. 2,490,496, as well as European Patent Application No. 0,049,182 describe immuno-stimulating glycoproteins extracted from Klebsiella pneumoniae wherein they contain 30$ to 45$ of proteins, 30% to 40$ of neutral carbohydrates, less than 4$ of glucuronic acid and 2$ to 5$ of osamines, and have a molecular weight of about 350,000 daltons. Particularly preferred of the above proteins are those wherein the protein fraction is composed of about % of acidic amino acids and in that the polysaccharide fraction contains approximately one glucose molecule per four galactose molecules and/or is essentially composed of repeating polysaccharide units, the structure of which is: ft ^ga lactose^ -Sgalactosel 'glucose CL Jn in which m is an integer 3, 4 or 5- The aforementioned applications also describe a process for preparing these glycoproteins, wherein a solution of glycoproteins obtained from an extract of cultures of Klebsiella pneumoniae (e.g. by diafiltration of an extract of a lysate of such cultures) is treated with a quaternary ammonium compound; the resulting precipitate is eliminated; the supernatant thus obtained, corresponding to a saline solution of the glycoproteins, is treated when cold with an alkanol of low molecular weight; the new precipitate thus obtained is dissolved in water, dialyzed. ,/ -2lyophilized, put back into solution and filtered on a gel; and the first eluted fraction is co] 1 r^-t »*o, '’oncent rat <‘d and, if desired, taken to dryness.

These applications also describe the immunoS stimulating activity of the said glycoproteins .ind t heir use in therapy, in particular present, <·.·υ in t.he form of pharmaceutical compositions.

Studies carried out since these applications were filed have enabled the structure of these glycoproteins to be clarified. Thus it has been found that these glycoproteins are more precisely acylgJycoproteins, the polysaccharide chain of which is linked to an asparagine residue of the protein chain by a core comprising heptose and 2-keto-3-deoxy-octulosonate, followed by an acyl part containing β-hydroxymyristic acid, then bv N-acety1-qlycosamine. These studies have also pnahled prominence to be given to the anti-allergic activity of the said glycoproteins.

By pursuing these studies also in the framework oi the process, the applicant has discovered that, it is possible to obtain these glycoproteins on an industrial scale more economically, bv concentrating the supernatant obtained after elimination of the precipitate arising from the action of the quaternary ammonium compound. Such a concentration is carried out, according to the present invention by use ol devices for the selection of molecules. These devices not only result in the possibility of using smaller volumes in the remainder of the process · but. also in an unforeseen manner, make it possible to dispense with the subsequent stages of dialysis and of tiltration on gel required in the previously described processes for obtaining these glycoproteins.

Thus according to one feature of the present invention there is provided a process tor obtaininq glycoproteins in which a solution of purified glycoproteins is prepared by diafiltration of an extract -3of a lysate of cultures of Klebsiella pneumoniae; the said solution is treated with a halide of a quaternary ammonium conpound; and the supernatant is isolated by elimination of the precipitateobtained, wherein in 5 the said process the supernatant is concentrated hy use of at least one device for the selection of molecules; the concentrate thus formed is treated when cold with at least one alkanol of low molecular weight; and the precipitate thus obtained is isolated, washed, if desired, with at least one alkanol of low molecular weight and dried.

This new process is surprisingly more effective than that of the previous art since it enables, in particular, two long and costly staqes to be dispensed with, as well as reducing the volume of alkanol used.

The above process, up to the obtaining of 1 the supernatant, may be carried out as described in the above-mentioned French and European applications.

The concentration may be effected by the usual methods ot selecting molecules, in particular by using one or more membranes of selective permeability, notably uJtrafiIters.

The membranes of selective permeability may 25 be of various kinds. They may be of cellulose acetate such as the membranes of HF-U (Dow Chemicals), HF-U (Kalle Chemie), SEPA-CA (Osmonics), or of SM (Sartorius) type. They may be complex polyelect rolytehascri, such as the membranes ot UM /Amicon) or of IRIS 3042 (Rhone-Poulene) type. They may also he polysulphono-based, such as the membranes of H10P (Amicon and Romicon), SEPA PS (Osmonics) or of IRIS 3022 (Rhone-Poulenc) type. They may also be polyamide-based such as the membranes of BM or BHF (Berqhof) type, aromatic polymer-based, such as the membranes of PM, MX or HF /Amicon and -4Romicon) type, copolymer-based vinyl chloride and acrylonitrile, based on substituted polyolefins or further composites based on polysulphones on polyethylene supports such as membranes of PT (Millipore) type.

These membranes may be presented in flat or tubular form or, further, in the form of hollow fibres or of spirals.

In order to obtain the desired acylglycoproteins 10 in pure form, ultrafilters are preferably used, the cut-off threshold of which is set at a molecular weight ranging from 5,000 to 100,000 daltons.

In the preferred conditions for putting the process into operation, ultrafilters are used which are presented in the form of hollow fibres, notably those of polysulphonic form with a cut-off threshold fixed at 5,000 daltons. Such ultrafilters include the membranes of H10P5 type, made by the Amicon and Romicon companies.

The operation of concentration is advantageously carried out in the region of ambient temperature, for example between 20-30°C. It can be carried out according to the techniques recommended by the maker of the membranes utilized. It is possible to use a single membrane or several different membranes and the operation of concentration may be carried out once or several times, in either a continuous or discontinuous manner.

When hollow fibres are used, the concentration is carried out preferably in two treatment cycles by using the wash-in technique which consists of compensating for the losses in the filtration chamber of small molecules and solvent by an intake of solvent (water in the case of the present invention) in the filtration chamber.

The concentration which it is desirable to obtain is about five times that of the solution at the start. -5The solution obtained is then treated when cold at about +4°C, with an alkanol of low molecular weight such as e.g. methanol, ethanol, n-propanol or isopropanol. Ethanol is preferably used.

The most useful results are obtained by using six volumes of ethanol per one volume of salt solution, overnight, at a temperature of +4°C.

Next, one or more techniques are used for isolating the precipitate, such as, for example, decanting followed by filtration, solely filtration, or centrifugation.

The precipitate may then advantageously be dried, at for example ambient pressure, but preferably under reduced pressure, in the presence or absence of a dehydrating agent.

As dehydrating agent, potassium hydroxide, phosphoric anhydride or, preferably, calcium chloride may be used, for example. Drying may be assisted by slight heating, preferably at a temperature less than 40°C. If desired, the precipitate may at this time be homogenized, for example by mechanical crushing.

The precipitate may also be dissolved in water and taken to dryness by, for example, atomization or lyophilization. Lyophilization is carried out in conventional manner, for example in freezersublimation units of average size as in the SMCJ or SMRG models marketed by Societ/ Usifroid, lyophilisators of large size as, for example, the unit formed by a CAl freezer and an SMIRS sublimator, both marketed by Usifroid. Smaller laboratory models may also be used, as well as those marketed by other companies such as Societe Serail. The extract for use as the starting material in the process according to the invention may be obtained, for example as indicated in French Patent Specification No. 2,171,907, that is to say, for example, by culturing Klebsiella pneumoniae (e.g. Klebsiella -6pneumoniae CtP 52145) preferably identical to the strain filed by the applicant on 29t.h June 1981 under Wo, 1-163 at the Institut Pasteur at Paris, The microorganisms are then lysed dried (e.g, by lyophilisation) and lipids may be extracted therefrom by means of solvents. The extract thus formed J may then be physically de-proteinised (by centrifugation, for example), then ultrafiltered and dried, for example by lyophilisation.

The glycoproteins as described in French Application No. 2,490,496, as well as in European Patent Application No. 0,049,182, and likewise those described in the French Patent Specification Wo. 2,171,907, which glycoproteins may advantageously be purified by the process according to the invention, are endowed with remarkable anti-allergic properties.

Thus, the present invention also has as its subject the glycoproteins obtained by the abovedescribed process according to the invention as well as the glycoproteins obtained by the process described in French Patent No. 2,171,907, for use in the treatment of allergic diseases as well as pharmaceutical compositions intended for the treatment of allergic diseases, wherein they contain such glycoproteins in association with a pharmaceutical excipient.

These pharmaceutical compositions may be, for example, solid or liquid and may be presented in the pharmaceutical forms currently used in human medicine for treating allergic diseases, such as for example tablets (plain or sugar-coated), capsules, syrups, aerosols, suppositories, injectable preparations, creams or ointments; they are prepared according , to the usual methods. The active principle(s) J 35 may be incorporated in the usual excipients used in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, -7fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting, dispersing or emulsifying agents, preservatives, colorants and flavourants.

Tbe glycoproteins and compositions which are the subject of the invention, find use in particular in the treatment of respiratory allergies such as allergic rhinitis and tracheitis and laryngitis, skin allergies such as eczema and urticaria, ocular allergies and allergic symptons of various origins such as e.g. animal stings and food allergies.

The following examples illustrate the invention without, however, limiting it.

I -8Example 1: At 4°C and over a period of J6 hours, L kg of product such as that obtained in Example 1 of French Patent Specification Ho. 2,171,907, prepared starting with the strain of Klebsiella pneumoniae 52145 or I 163 of the collection at the Institut Pasteur is put into aqueous solution at 10 g/litre. 0.8 volume of a 3% cetyltrimethylammonium bromide solution is added at a rate of about 1 litre/minute, and the whole is gently agitated for 1 hour. The precipitate formed is eliminated by continuous centrifugation at 62,000 g at a rate of about 5 litres/ hour.

The supernatant is concentrated by ultrafiltration on hollow fibres at a retention threshold fixed at 5,000 (Hollow Fibers H10P5 marketed by Amicon and Romicon) in two treatment cycles, in proportions of 5/1. At a speed of 3 litres per minute, 6 volumes of 96% ethanol is added and gently agitated for minutes. After decanting, separating, rinsing the precipitate, drying at less than 40°C in the presence of a hydrating agent and homogenizing by mechanical crushing, 200 g of the product sought is obtained, possessing the characteristics of the product described in French Patent Application Ho. 2,490,496, as well as in European Patent Application No. 0,049,182.

Example 2: Tablets are prepared corresponding to the formulation: - Product of Example 1............................. 1 mg - Excipients q.s. for a tablet finished at ........ 100 mg (detail of excipient: lactose, starch, talc, magnesium stearate).

Example 3: Aerosols are prepared releasing doses each containing: -9- Product of Example 1........................... 0.5 mg - Emulsifier .................................... 0.15 mg - Propellant .................................... r>0 mg Example 4s A cream is prepared corresponding to the t formulation: - Product of Example 1........................... 1 mg - Excipient: 2-octyl-dodecanol, cetostearyl * alcohol, sodium cetostearyl sulphate, methyl and propyl parahydroxybenzoate, purified water .................................10 g Example 5: Tablets are prepared corresponding to the formulation: - Product of Example 1 of French Patent Specification No. 2,171,907 .................................. 1 mg - Excipient q.s. for a tablet finished at .......100 mg (detail of excipient: lactose, starch, talc, magnesium stearate).

ANTI-ALLERGIC ACTIVITY Pr inciple Allergic subjects synthesise antibodies (Immuno globulins E or IGE) against the allergen to which they are sensitive. These antibodies are able to fix themselves on to the basophilic polynuclears (or granulocytes) of these subjects and lead to a modification of the membrane of the polynuclears which causes their de-granulation.

Test The technique is inspired by that of Benveniste Chim. Allergy., 1981, 11, p. 1 to 11. A plasma enriched with basophilic polynuclears from a subject sensitive to a given allergen is prepared by decanting, X starting with 1 part blood and 9 parts colorant (May-GrQnwald-Giensa). The control-suspension and the suspension which has had the product under test added to it are put in contact with the same allergen. The de-granulation is then evaluated -10as a percentage: No. of basophils of _ No. of basophils of %- the control suspension the treated suspension No. of basophils of $ the control suspension An anti-allergic agent reduces the percentage of de-granulation of the basophilic polynuclears. Results 1. Allergen : cat fur.

At a concentration of 100 ^jg of the product of Example 1 per ml., no de-granulation is observed in the treated suspension, whilst there is 61% de-granulation in the control suspension. 2) Allergen : whole mosquito body.

At concentrations of 2 ^jg of the product of Example 1 or of the starting product of Example 1, per 1 ml,, no de-granulation is observed in the treated suspension, whilst there is 60% degranulation in the control suspension.

Claims (12)

1. A process for obtaining glycoproteins in which a solution of purified glycoproteins is prepared by diafiltration of an extract of a lysate of cultures of Klebsiella pTieumoniae; the said solution is treated with a halide of; a quaternary ammonium compound; and the supernatant is isolated by elimination of the precipitate thus obtained, Wherein in the said process the supernatant is concentrated by use of at least one device for the selection of molecules; the concentrate thus formed is treated when cold with at least one alkanol of low molecular weight; and the precipitate thus obtained is isolated, washed, if desired, with at least one alkanol of low molecular weight and dried,

2. A process according to claim 1 wherein the device for the selection of molecules includes one or more membranes of selective permeability.

3. A process according to claim 1 or claim 2, wherein the device for selection of molecules comprises one or more ultrafilters.

4. A process according to claim 3 wherein the ultrafilters have a cut-off threshold calibrated to a molecular weight ranging from 5,000 to 100,000 daltons.

5. A process according to claim 3 or claim 4 wherein the ultrafilters are in the form of'hollow f ibres.

6. A process according to claim 5 wherein the ultrafilters have a polysulphonic form and are calibrated to 5,000 daltons.

7. A process according iao claim 1 substantially as herein described -12A process according to claim 1 substantially as herein described with reference to the Examples, g. Glycoproteins whenever obtained by a process > according to any one of claims 1 to

8. 5 10. Glycoproteins according to claim

9. For use in the treatment ot allergic diseases. e Glycoproteins obtained by lysing Klebsiella pneumoniae, drying, extracting lipids therefrom by means of solvents, physically de-proteinising,

10. Ultrafiltering and drying, for use in the treatment of allergic diseases.

11. 12. Pharmaceutical compositions comprising, as active ingredient, glycoproteins as claimed in claim 9 or claim 11 in association with a pharmaceutical 15 excipient.

12. 13 Pharmaceutical compositions as claimed in claim 12 for use in the treatment of allergic diseases.