NZ206959A - Preparation of immuno-stimulating acylglycoproteins extracted from klebsiella pneumoniae and 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

New Zealand Paient Spedficaiion for Paient Number £06959 \ 20695 9 Priority Date(s): .. T*? . i Complete Specification fiteu Class: %&/£>%.. mftz/99.., ! Ml fa 3/9. A * Publication Date: ?. .1 ...

; P.O. Journal, No: d nodrawikss Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION "Chemical Process" ¥£WE , ROUSSEL-UCLAF, a French Body Corporate of 35 Boulevard des Invalides, 75007 Paris, France, hereby declare the invention, for whichXR/we pray that a patent may be granted toxoe/us, and the method by which it is to be performed, to be particularly described in and by the following statement:- (fo!lowed by page I A.) 206959 - la- The present invention relates to a process for the preparation of immuno-stimulating acylglyco-proteins extracted from Klebsiella pneumoniae as well as to anti-allergic pharmaceutical compositions 5 containing acylglycoproteins prepared by such a process. ftrcnch Patent Application No: 2^490/496/ 6a well a0 European Patent Application No. 0,049,182 describe immuno-stimulating glycoproteins extracted 10 from Klebsiella pneumoniae characterised in that 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 15 preferred of the above proteins are those characterized in that the protein fraction is composed of about 30% of acidic amino acids and in that the polysaccharide fraction contains approximately one glucose molecule per four galactose molecules and/or is essentially 20 composed of repeating polysaccharide units, the structure of which is: Sgalactose1)^—:'galactose1—''glucose1^ in which m is an integer 3, 4 or 5. The aforementioned applications also describe a process for preparing 25 these glycoproteins, characterized in that 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 30 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 FEB 1987" thus obtained is dissolved in water, dialyzed, r- 206 9 5 Q lyophilized, put back into solution and filtered on a gel; and the first eluted fraction is collected, concentrated and, if desired, taken to dryness.

These applications also describe the immuno-5 stimulating activity of the said glycoproteins and their use in therapy, in particular presented in the form of pharmaceutical compositions.

Studies carried out since these applications were filed have enabled the structure of these 10 glycoproteins to be clarified. Thus it has been found that these glycoproteins are more precisely acylglycoproteins, the polysaccharide chain of which is linked to an asparagine residue of the protein chain by a core comprising heptose and 15 2-keto-3-deoxy-octulosonate, followed by an acyl part containing S-hydroxymyristic acid, then by N-acetyl-glycosamine. These studies have also enabled prominence to be given to the anti-allergic activity of the said glycoproteins. 20 By pursuing these studies also in the framework of the process, the applicant has discovered that it is possible to obtain these glycoproteins on an industrial scale more economically, by concentrating the supernatant obtained after elimination of the 25 precipitate arising from the action of the quaternary ammonium compound. Such a concentration is carried out, according to the present invention by use of devices for the selection of molecules. These devices not only result in the possibility of using 30 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 filtration on gel required in the previously described processes for obtaining these glycoproteins. 35 Thus according to one feature of the present invention there is provided a process for obtaining glycoproteins in which a solution of purified glycoproteins is prepared from an extract of cultures 206959 of Klebs iella pneumoniae (e.g. 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 compound; and the supernatant is isolated by elimination of the precipitate thus obtained, the said process being characterized in that the supernatant is concentrated by use of at least one device for the selection of molecules; the concentrate thus formed is treated when cold c,_c3 with at least oneyalkanol cff low molecular weight*; and the precipitate thus obtained is ^.isolated, washed, if desired, with at least one'al^anol afr flow molcculac weight/ and dried.

This new process is surprisingly more effective 15 than that of the previous art since it enables, in particular, two long and costly stages to be dispensed with, as well as reducing the volume of alkanol used.

The above process, up to the obtaining of the supernatant, may be carried out as described sptcijtca&o/i in the above-mentioned French ana European Applications.

The concentration may be effected by the usual methods of selecting molecules, in particular by using ore or more membranes of selective permeability, 25 notably ultrafilters.

The membranes of selective permeability may 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 30 SM (Sartorius) type. They may be complex polyelectrolyte-based, such as the membranes of UM (Amicon) or of IRIS 3042 (Rhone-Poulenc) type. They may also be polysulphone-based, such as the membranes of H10P (Amicon and Romicon) , SEPA PS (Osmonics) or 35 of IRIS 3022 (Rhone-Poulenc) type. They may also h Ny be polyamide-based such as the membranes of BM £■'<*. f/.\t or BHF (Berghof) type, aromatic polymer-based, If tj such as the membranes of PM, MX or HF (Amicon and | " ^FBBi98, V* ■ - Romicon) 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) 5 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 15 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 25 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 30 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) 35 in the filtration chamber.

The concentration which it is desirable to obtain is about five times that of the solution at the start. 206959 The solution obtained is then treated when o $e/e.c-&6: 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, 10 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 15 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 20 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 freezer- sublimation units of average size as in the SMU or SMRG models marketed by Societ^ Usifroid, lyophili- sators 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, 6ri -hsh. ' for example as indicated in French Patent Specification cl, No. p, 171,OOf, that is to say, for example, by +FEB1987Z culturing Klebsiella pneumoniae (e.g. Klebsiella / r-- 20695 The 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 freezer-sublimation units of average size as in the SMU or SMRG models marketed by Societe^ Usifroid, lyophili-sators 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 20bS50 pneumoniae CIP 52145) preferably identical to the strain filed by the applicant on 29th June 1981 under No. 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 may then be physically de-proteinised (by centrifugation, for example), then ultrafiltered and dried, for example by lyophilisation.

The glycoproteins as described in frrcncK Application No.—2,490,496 f—ao well ao it/ European Patent No. 0,049,182, and likewise British those described in the French Patent Specification No. 12,171,907, which glycoproteins may advantageously 15 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 above-described process according to the invention as 20 well as the glycoproteins obtained by the process dr/tisA JOil . Suila-fifG. described in fPecncn 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, characterized in that they 25 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 30 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) may be incorporated in the usual excipients used in these pharmaceutical compositions, such as talc, fc /V gum arabic, lactose, starch, magnesium stearate, // s 0\ cocoa butter, aqueous or non-aqueous vehicles, 695 fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting, dispersing or emulsifying agents, preservatives, colorants and flavourants.

The 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 10 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. 206959 Example 1: At 4°C and over a period of 16 hours, 1 kg of product such as that obtained in Example 1 of French Patent Specification No. tt, 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 15 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 fronoh Patent Application? Sp 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 ae prepared releasing doses each ^ ^ q containing: /v ^ \-4FEB1987£; \<*. /> 206353 - Product of Example 1... 0.5 mg - Emulsifier 0.15 mg - Propellent 50 mg Example 4: A cream is prepared corresponding to the 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: . 6rit»s h - Product of Example 1 of yronoh Patent Specification ht+ia. zoa No. p, 171,007 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 25 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, starting with 1 part blood and 9 parts colorant 35 (May-GrOnwald-Giensa) . The control-suspension and the suspension which has had the product under £^ /' c' test added to it are put in contact with the same/V z allergen. The de-granulation is then evaluated ( m4FEB198/ o, I V £

Claims (16)

as 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 jig 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 ^g 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% de-granulation in the control suspension. 206959 WHAT -T/WE CLA'M 15.- Olaima " •%

1. A process for obtaining glycoproteins in which a solution of purified glycoproteins is prepared from an extract of cultures of Klebsiella pneumoniae; 5 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, the said process being characterized in that the supernatant is concentrated by use 10 of at least one device for the selection of molecules; the concentrate thus formed is treated when cold c,-c3 with at least one^alkanol ^f low molecular weight; and the precipitate thus obtained is isolated, washed, if desired, with at least one^alkanol.^erf 15 )tow molooular 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, 20 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 25 daltons.

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

6. A process according to claim 5 wherein the 30 ultrafilters have a polysulphonic form and are calibrated to 5,000 daltons.

7. A process as claimed in any one of the preceding claims wherein the initial solution of purified glycoproteins is prepared by diafiltration of an 35 extract of a lysate of cultures of Klebsiella pneumoniae.

8. A process according to claim 1 substantially^'^f^^^ as herein described. f/V ■<<-. -4FEBI987Z - 12 - 206959

9. A process according to claim 1 substantially as herein described with reference to the Examples.

10. Glycoproteins whenever obtained by a process according to any one of claims 1 to 9. Su,icU>ie.

11. Glycoproteins according to claim lO^for use in the treatment of allergic diseases.

12. Glycoproteins obtained by lysing Klebsiella pneumoniae, drying, extracting lipids therefrom by means of solvents, Phys^ica^^ de-proteinising, ultrafiltering and drying,^for use in the treatment of allergic diseases.

13. Pharmaceutical compositions comprising, as active ingredient, glycoproteins as claimed in claim 10 or claim 12 in association with a pharmaceutical excipient.

14. Pharmaceutical compositions as claimed in SuitcLbli. claim 13/for use in the treatment of allergic diseases. in asKmoJs o&tzf tt*as\ kuyrtans

15. Method of treatment of allergic diseases^ characterized in that at least one of the glycoproteins as defined in claim 10 is used.

16. Method of treatment of allergic diseases^ characterized in that at least one of the glycoproteins as defined in claim 12 is used. -ir?-: Bach and every novel method/ procooo, composition and product heroin disclosed,- BALDWIN, SON £ CAREY attorneys for the applicants ~4FEB1987£ ■ // I V