FI78304B - NYTT FOERFARANDE FOER FRAMSTAELLNING AV KLEBSIELLA PNEUMONIA EXTRAHERADE IMMUNSYSTEMET STIMULERANDE ACYLGLYKOPROTEINER. - 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

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The present invention relates to a novel process for the preparation of immune-stimulating acyl glycoproteins extracted from Klebsiella pneumoniae.

FR Patent No. 2,490,496 and European Patent No. 0,049,182 describe 10 novel immune system-stimulating glycoproteins, especially those extracted from Klebsiella pneumoniae, which are characterized by containing 30-40% proteins, 30-40% neutral sugars , up to 4% glucuronic acid, 2-5% amino sugars and having a molecular weight of about 350,000 daltons. In particular, they describe the proteins described above, characterized in that the protein fraction consists of about 30% acidic amino acids and that the polysaccharide fraction contains on average one glucose molecule per 4 galactose molecules and consists essentially of repeating polysaccharide units, having the following structures: 13 14 1 - [(galactose) -— galactose-glucose where m is an integer of 3,4 or 5. These 25 patents also describe a process for the preparation of these β-glycoproteins, characterized in that the resulting glycoprotein solution is treated with a quaternary ammonium compound, ultrafiltrating the extract from the lysates of Klebsiella pneumoniae cultures, separating the corresponding supernatant by removing the precipitate obtained, treating the supernatant corresponding to the saline solution of glycoproteins with a small alkanol in the cold to obtain new precipitates which are dissolved in water, dialyzed, lyophilized, dissolved ethane, gel filtration, recovery of the first eluted fraction, concentration and, if desired, drying.

These patents also describe the immune system stimulating effect of said glycoproteins and their therapeutic use, in particular in the form of pharmaceutical preparations.

Studies performed since the registration of these patents have made it possible to elucidate the structure of these glycoproteins. These are specifically glycoproteins, but more specifically acyl-10 liglycoproteins whose polysaccharide chain is bound to the asparagine residue of the protein chain at a site formed by heptose and 2-keto-3-deoxyoctylosonate followed by an acylated moiety formed by ^ -h From N-acetyl-15 glucosamine. These studies have also made it possible to demonstrate the antiallergic effect of these glycoproteins.

The studies presented in the method also show that it was possible to take better account of the economic requirements of carrying out an industrial process by concentrating the supernatant recovered after removal of the precipitate obtained using a quaternary ammonium salt. This concentration is preferably performed using devices with a selectively permeable membrane, such as ultrafiltration devices, to separate the molecules. In fact, these devices make it possible, on the one hand, to process smaller volumes in the later stages of the process and, on the other hand, to surprisingly access the dialysis and gel filtration steps finally completed in the previous process.

Thus, the present invention relates to a new process for the preparation of glycoproteins described in FR Patent No. 2,490,496 and in EP Patent No. 0,049,182.

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More particularly, the invention relates to a process for the preparation of glycoproteins by preparing a purified glycoprotein solution by ultrafiltration of a lysate extract of Klebsiella Penu moneae cultures, treating the resulting solution with a quaternary ammonium halide and separating the supernatant by removing the precipitate thus obtained. filter to separate the molecules, the concentrate is treated cold with at least one lower alkanol, and that the precipitate thus obtained is separated and, if necessary, washed with at least one lower alkanol and dried.

This new method is surprisingly better than the previous method, as it allows in particular 15 of the two long and expensive steps and can reduce the volumes of alkanol to be treated.

Concentration can be performed by conventional Mole-separation methods, in particular by using membranes with selective permeability, especially ultrafilters. 20 Selectively permeable films may be different in nature. They can be cellulose acetate such as well types HF-U (Dow chemicals), HF-U (Kalle Chemie), SEPA-CA (Osmonics) or SM (Sartorius). They can be polyelectrolyte-based, such as well types UM 25 (Amicon) or IRIS 3042 (Rhone-Poluenc). They can also be polysulfone-based such as well types H10P (Amicon and Romicon), SEPÄ PS (Osmonics) or IRIS 3022 (Rhone-Poulenc). They can also be polyamide-based, such as well types BM or BHF (Berghof), aromatic polymers such as PM, MX or HF (Amicon or Romicon), vinyl chloride and acrylonitrile based copolymers, substituted polyolefins such as polyolefins or even mixed forms, well types PT (Millipore).

35 These films can be in the form of planes, tubes or even *. ; as hollow fibers or spirals.

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In order to obtain the desired acyl glycoproteins in pure form, ultrafilters with a filtration threshold calibrated for molecular weights that can range from 5,000 to 100,000 daltons are preferably used.

5 Under the best conditions for carrying out the process, ultrafilters are used, in particular as polysulphone-based, hollow fibers with a filtration threshold of 5000 daltons.

Thus, film types H10P manufactured by Amicon and Romicon are preferably used.

The concentration is preferably carried out at about room temperature, for example at 20-30 ° C. It can be performed using the technique recommended by the manufacturer of the films used. It is possible to use a single film or several different films, and the concentration can be carried out once or more continuously or intermittently.

When hollow fibers are used, the concentration is preferably performed in two treatments using the "Wash in" technique, replacing the liquid leaving the filtration chamber with small molecules and dissolving by adding solvent (water in the case of this invention) to the filtration chamber.

The desired concentration is about 5 times the starting; compared to the solution.

The resulting solution is then treated in the cold at about 25 ° C with a low molecular weight alkanol such as methanol, ethanol, n-propanol or isopropanol.

Ethanol is preferably used.

The most interesting results have been obtained using five volumes of ethanol per volume of saline 30 while standing overnight at + 4 ° C.

Several techniques are then used to isolate the fines, such as decantation, followed by filtration, filtration alone, or centrifugation.

The precipitate can then preferably be dried, for example at normal pressure, but preferably at reduced pressure, with or without a dehydrating agent. As the dehydrating agent, for example, potassium hydroxide, phosphoric anhydride or, preferably, calcium chloride can be used. Drying can be promoted by heating, preferably below 40 ° C. If desired, the precipitate can be homogenized at this stage, for example by mechanical grinding.

The precipitate can also be dissolved in water and evaporated to dryness, for example by killing or lyophilization.

Lyophilization is then performed by a conventional conventional method, for example a medium-sized combination of freezing and fermenting machines such as SMU or SMRG models marketed by Usifroid, large lyophilizers such as a device consisting of CAI freezer and SMIRS fermenter, both of which are marketed.

Smaller labs lie and can also be used as well as equipment marketed by other companies such as the Serail Company.

The starting materials for the process of the present invention can be prepared as in FR Patent No. 2,171,907, i.e. by culturing bacteria (Klebsiella pneumoniae CIP 52145), which are the same as strain 1-163 deposited by the applicant with the Pasteur Institute in Paris on June 29, 1981. by disrupting the bacteria, drying (for example by lyophilization), extracting with lipid solvents, physically removing the proteins (for example by centrifugation), then ultrafiltrating and drying, for example by lyophilization.

When shaped with a suitable filler, these glycoproteins have, as described in the FR patent: ·. European Patent No. 2,490,496 and European Patent No. 0 049 182 and FR Patent No. 2,171,907 have significant antiallergic properties.

The glycoproteins described and patented in 35 FR Patent No. 2,490,496 and European Patent No. 6,780,302 and described in U.S. Patent No. 2,171,907 can thus be used for the preparation of pharmaceutical preparations intended for for the treatment of allergic diseases.

These pharmaceutical preparations may be e.g.

in solid or solution and pharmaceutical forms commonly used in human medicine for use in the treatment of allergic diseases, such as, for example, uncoated or coated tablets, capsules, syrups, aerosols, suppositories, injectables, ointments, ointments; they can be prepared by conventional methods. The active ingredient (s) may be in admixture with excipients commonly used in pharmaceutical preparations such as talc, acacia, lactose, starch, magnesium stearate, cocoa butter, anhydrous or aqueous carriers, vegetable or animal fats, paraffins, paraffins various blowing agents, dispersants or emulsifiers, preservatives, colorants, flavors.

The preparations are used in particular for the treatment of respiratory allergies such as allergic rhinitis, inflammation of the trachea or larynx, subcutaneous allergies such as rash or urticaria, eye allergies or allergies caused by various causes: for example, animal stings or food allergies.

The following examples illustrate the invention.

Example 1

Dissolve in water at 4 ° C for 16 hours at a concentration of 10 g / l 1 kg of the same compound prepared in Example 1 of FR Patent No. 2,171,907 obtained using Klebsiella pneumoniae 52145 or I 163 as starting material. positions on the collections of the Pasteur Institute. Add 0.8 volumes of a 3% solution of cetyltrimethylammonium bromo-35 midi at about 1 liter / min. and stir moderately for one hour. The precipitate formed is continuously removed by centrifugation at a flow rate of about 5 liters / min at 62,000 g.

The supernatant is concentrated by ultrafiltration through hollow fibers with a retention threshold of 5000 5 (Hollow fiber H10P5 sold by Amicon and Romicon) in two rounds of treatment at 5/1 ratios. At a rate of 3 liters per minute, 6 volumes of 96% ethanol are added and stirred moderately for 15 minutes. Allow to settle, centrifuge and rinse the precipitate, dry below 40 ° C in the presence of a dehydrating agent, homogenize by mechanical grinding to give 200 g of the desired compound having the properties of the compound described in FR patent n: o 2,490,496 and European Patent 15 No. 0 049 182.

Example 2

Tablets were prepared having the composition of: The compound of Example 1 1 mg Excipient ad 100 mg 20 (Excipient in particular: lactose, starch, talc, magnesium stearate).

Example 3

Aerosols were prepared, the next dose of which contained: 0.5 mg of the compound of Example 1

Emulsifier 0.15 mg

Propellant 50 mg

Example 4

A fat was prepared having the following composition: Compound of Example 1 1 mg Excipient: 2-octyldodecanol, ketostearyl alcohol, sodium ketostearyl sulfate, methyl and propyl parahydroxybenzoate, purified water 10 g.

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Example 5

Tablets were prepared having the composition of: Compound of Example 1 of FR Patent No. 2,171,907 1 mg 5 Filler ad 100 mg (Filler specifically: lactose, starch, talc, magnesium teaate).

Antiallergic effect

People with allergies synthesize antibodies 10 (Immunoglobulin E or IgE) to the allergen to which they are sensitized. These antibodies are able to bind to the surface of the basophils of this human polynuclear (or granulocytes) and cause a change in the membrane of the polynuclear cells that causes them to degranulate.

Test:

The technology comes from Benvensiste Chim. Allergy, 1981, 11, pp. 1-11. Prepared by decanting a plasma rich in polynuclear basophils from 20 individuals who have been sensitized to a particular allergen by taking one part blood and 9 parts dye (May-Griinwald-Giemsa). The control suspension and the suspension to which the test substance has been added are reacted with the same allergen. Then determine 25% degranulation:

Control suspension basophils Number of treated suspension _ number _ sio.i basophiles _number_ '

Number of basophils in control suspension

The antiallergic agent reduces the percentage of degranulation of polynuclear basophils.

Results: 1) Allergen: cat hair

At a concentration of 100 μg of the compound of Example 1 in 1 ml, no degranulation is observed in the treated suspension, while 61% degranulation is observed in the control suspension.

9 78304 2) Allergen: mosquito whole At a concentration of 2 μg of the compound of Example 1 or the starting material of Example 1 in 1 ml, no degranulation is observed in the treated suspension, whereas 60% degranulation is observed in the control suspension.

Claims (6)

10 78304 A process for the preparation of glycoproteins by preparing a purified glycoprotein solution by ultrafiltration on a lysate extract of Klebsiella pneuomiae cultures, treating the resulting solution with a quaternary ammonium halide and separating the supernatant by concentrating the supernatant, separating the supernatant using with at least one lower alkanol, and that the precipitate thus obtained is separated, washed, if necessary, with at least one lower alkanol and dried. A method according to claim 1, characterized in that the ultrafilter comprises one or more membranes with a selective permeability. Method according to Claim 1 or 2, characterized in that several ultrafilters are used. A method according to claim 3, characterized in that the ultrafilters have a filtration threshold calibrated for molecular weights of 5,000 to 100,000 daltons. Method according to Claim 3 or 4, characterized in that the ultrafilters are in the form of hollow fibers. Method according to Claim 5, characterized in that the ultrafilters are polysulfone-based and are calibrated to 5000 daltons. li