DK146724B - PROCEDURE FOR THE PREPARATION OF SLOW ALFA AND BETA GLYCOPROTEINS - Google Patents

Description

U6724

The invention relates to a process for the preparation of slow α and β glycoproteins as set forth in the preamble of claim.

Danish Patent Specification No. 129,466 discloses such a method for extracting previously light-treated cultures of microbial strains.

This process is thus: - cultivating certain microbes and extracting the microbes, - making a lysis of them, - subjecting the residual effect of one or more lipid solvents and liberating the obtained solution for proteins by physical or chemical means, - selectively, from the protein-free residue, the slow glycoproteins are precipitated by the addition of a solvent or a mixture of solvents.

The glycoproteins obtained by this method have very interesting therapeutic properties. They carried significant anti-inflammatory properties and rapid and nonspecific immunization properties. They also have the advantage of not being allergic or hyperthermizing, nor of causing intolerance phenomena at the injection site.

The structure of these slow α and β glycoproteins is demonstrated by their electrophoretic behavior in density gradient relative to reference glycoproteins, especially in relation to serum glycoproteins,

The chemical nature of these substances is demonstrated by chemical reactions, such as their content of hexoses and pentoses, by approximating the molecular weight using selective chromatography agents such as modified celluloses, sephadex gel ("Sephadex" is a registered trademark for polymerized dextran) or molecular sieves, of protein fraction judged by the biurethogenic ability calculated relative to a serum albumin standard, by the content of hexosamines, and by the content of sialic acid.

The mixture of slow α and β glycoproteins * obtained by this process can be defined as consisting of slow, thermostable, acid soluble and ammonium sulfate solutions soluble α and β glycoproteins.

2 146724

The slow ct and β-glycoproteins obtained by said process have a combined hexose content between 50 and 60 $.

The process of the invention for the preparation of slow α and β glycoproteins, as further specified in the preamble of claim 1, is characterized by the characterizing part of the claim.

The process of the invention makes it possible to obtain slow α and β glycoproteins in a very pure and consequently very active form. The process makes it possible to eliminate most of the degradation products from the proteins which have yet been able to remain in the mixture, and it is therefore possible to obtain the final product in a widely purified form.

The diafiltration on one or more calibrated membranes is unknown.

The pharmacological results given below show that, unlike a simple purification, the diafiltration produces a surprising result. In the case of a simple purification where the inactive products are eliminated, the activity of the products in question will be multiplied by the same ratio in the various experiments relative to the compounds of the prior art.

This is j. in fact, while the activities of the prior art substances and methods of the present invention are practically identical in the tests for simulating the specific defense mechanisms and antibacterial activity, the compounds prepared by the method of the present invention are, on the contrary, twice as active. in the test for anti-inflammatory effect.

The process of the invention is particularly distinguished by the following points: 1) The mesh shaped selectively permeable membranes are preferably those sold under the designation "Amicon XM 500" by Amicon Corporation, Lexington, Mass., USA.

2) The selective permeability membranes are preferably those sold under the brands "Amicon" XM 50, PM 30 and UM 2. The XM 300 membrane retains substances whose molecular weight is above 960,000, the membrane XM 50 retains s, if the molecular weight is above 142,000, the membrane PM 30 retains substances whose molecular weight is above 64,000, and the membrane UM 2 retains substances whose molecular weight is above 12,400, 3) the hydrophilic polymerized gel is a gel of "Sepharese" 6B, (registered trademark) .

4) The hydrophilic polymerized gel is a gel of "Sephadex" G 200. (registered trademark).

The slow α and β glycoproteins obtained by the process of the invention are defined by their molecular weight, their content of combined hexoses and by the ratio: combined hexose proteins

The very pure fraction obtained by this process is made up of glycoproteins having a molecular weight of or above 10 6 daltons containing between 60 and 65 $ combined hexoses and having a ratio of hexose protein substances in the vicinity of 7. It usually contains about $ 62 combined hexoses. Nevertheless, due to the difficulty of standardizing the measurement of sugars, this content may range between $ 60 and $ 65 without the possibility of drawing conclusions regarding the purity of the fraction.

The slow α and β glycoproteins obtained by the method of the invention are useful as anti-inflammatory agents and as agents for stimulating the specific defense of the organism. They are particularly suitable for the treatment of osteoarticular disorders, bronchitis and microbial infections.

They can be used as active ingredient in pharmaceutical compositions for administration by parenteral, perlingual, oral or rectal route or via the mucosa or in topical application. For example, injectable solutions or suspensions, conditioned in ampoules or multi-dose vials, lozenges, enteral sheath tablets, aerosols, nebulizers, cream, pomade, lotions, suppositories or vaginal suppositories may be mentioned.

The useful dose may vary widely depending on the therapeutic indication and the method of administration.

The following example illustrates the method of the invention.

Preparation of starting material.

1) preparation of bacterial extract.

The selected bacterial strain (Klebsiella pneumoniae) is grown in a culture vessel in an appropriate environment. Upon completion of cultivation, the germs are subjected to light for 8 days until stability of the bacterial suspension.

the lysate is then evaporated and freed of lipids by a mixture of methylal and methanol in the ratio of 4 µl. The centrifuge bottom is washed with methanol, taken up in water and added to formol. The bacterial extract is subjected to a final purification by precipitating its aqueous solution with the mixture of methylal and methanol and washing the precipitate with methanol.

2) Properties of the bacterial extract.

The resulting extract is clear and soluble in aqueous environment and does not precipitate in a 10 # (w / v) trichloroacetic acid environment, in 0, JN perehydrochloric acid or in 3.6 M ammonium sulfate solution at a pH of 7.0 and at 15 ° C.

Partial precipitation is obtained by the addition of a solution of manganese sulphate or cetylpyridinium chloride (C.P.C.).

The major constituents of the bacterial extract have been determined, and the dosages have in particular indicated a content of combined neutral hexoses of 26-32 # depending on the charges and 6.2 # α-aminated nitrogen.

Example.

Dissolve 2 g of slow α and β glycoproteins obtained by the procedure described above from a culture of Klebsiella pneumoniae in 400 ml of water. This solution is introduced into a dialysis cell which is closed with a membrane of "Amicon" XM 300. The solution is bubbled under constant stirring and under slight pressure (0.7 bar). Dialysis is performed while still shifting the dialysis equilibrium by replacing the filtered volume. To this end, an equal volume of distilled water is added. This supply is continuous and the diafiltration operation is considered completed when the total diafiltered volume is equal to or greater than 10 times the initial volume. The residue in the diafiltration cell is collected and gel filtered on "Sepharose" 6B, and the fraction which is not fixed on the gel of "Sepharose" 6B is collected and dried by lyophilization.

You can also work in several steps, first performing a diafiltration in a dialysis cell with a membrane of "Amicon" PM 30 and then in a second dialysis cell with a membrane of "Amicon" X 50 and finally a third diafiltration in a cell with a membrane of "Amicon" XM 300. After gel filtration on "Sepharose" 6B and lyophilization of the unfixed fraction, a fraction is obtained which is virtually identical to that obtained after a single diafiltration on membrane.

The slow α and β glycoproteins thus obtained (retained on a membrane of XM 300 and not retained but excluded by a gel of "Sepharose" 6B) have a molecular weight equal to or greater than 10 6 daltons. These macromolecular substances have a composition with respect to hexoses which is better than the composition of the less pure fractions obtained by the method of the above-mentioned patent, the content usually being in the vicinity of 62 #.

In contrast, the content of α-aminated nitrogen is small (1.6 #) and less than the content of the previously prepared fractions.

The slow α and β glycoproteins thus obtained are dissociated only partially by urea in 8 M solution or by solutions of reducing agents. In contrast, the low molecular weight molecules that are separated by the diafiltrations can be spontaneously reassociated when the dissociation agent is eliminated. Do the thus obtained "de novo" macromolecules differ from the slow α and β glycoproteins retained on the membrane of "Amicon" XM 300; by subjecting the slow α and β glycoproteins to the effects of various enzymes (pronase, trypsin, pan-creatine and hyaluronidase) followed by a fractionation on membranes, Iran finds that a small proportion of the macro-molecules are sensitive to these enzymes and that the pharmacological activity is not appreciably modified.

The yield of the purification according to the working method described above is 30-35. The slow ce and β-glyoproteins thus obtained contain approx. 62? 6 combined hex-oes and approx. 9fo protein substance and the ratio of combined hexoses of protein substances is close to 7. Nevertheless, the interpretation is made difficult by the fact that the standardization of orcinol staining does not occur with the sugars actually contained in the slow a and β glycoproteins, but arbitrarily by means of galactose and mannose.

Structure Study ♦

Acidic hydrolysis under vacuum makes it possible to ascertain the various constituents of the molecules after separation. cellulose plate. Thus, a very large difference in amino acids can be found, which separates the slow α and β glycoproteins from the peptidoglycans, as well as osamines. The presence of glucose, galactose and mannose is observed among the sugars.

Presence of ribose is not detected.

Following alkaline hydrolysis, which causes breakage of the O-osidyl bonds, examination of the fragments formed shows that the long glycan chains are grafted onto small protein masks.

Thus, there is a molecule of glycoprotein nature which is demonstrated by its macromolecular nature, by its resistance to proteolytic enzymes and by its resistance to lipolytic enzymes (lipase and pancreatin). Furthermore, it is not a mueopolysaccharide,. since it is not precipitated by cetylpyridinium chloride.

In particular, electrophoresis in density gradient at a pH of 8.2 in the presence of a sodium barbital buffer shows a peak of (α-β) mobility.

pharmacological study of the glycoproteins. a) Anti-inflammatory effect.

To study the anti-inflammatory effect, the sample is used with plantar edema, which is triggered by local injection of various phlogogens, ie. tissue-altering agents, each of which elicits a slightly different inflammatory manifestation.

The edema is measured by plethysmometry before and after the injection of the phlogogenic agent.

The tested compounds are administered intraperitoneally 1 hour before the injection of the irritant. The active dose is determined in mg / kg which causes a $ 40 regression of the edema compared to control animals.

available results obtained with slow g and β-glycoproteins prepared according to the method of Danish Patent Publication No. 129 -466

Phlogogenic agent Active dose 40 #

Oarragenin 0.05 mg Ag

Kaolin 0.5 mg Ag

Ovalbumin 1 mgAg

Hemolysin 0.2 mgAg

Comparative result is between the slow oc and g-glycoproteins prepared according to the method of Danish Patent No. 229. ^ 66 and designated glvcoproteins A and the slow q and g-glyvo proteins made by the method of the present invention and denoted gl. protein proteins B.

A comparison is made for a podal edema 8 produced by carrageenin between the glycoproteins A and glycoproteins B and the most commonly used experimental anti-inflammatory agents:

Antidepressants Active dose $ 40

Indomethacin 2 mg / kg

Deltacortisone 5 mg / kg

Phenylbutazone 50 mgAg

Glycoproteins A 0.05 mgAg

Glycoproteins B 0.025 mgAg

The glycoproteins A and, in particular, the glycoproteins B are found to be the most potent experimental anti-inflammatory agents. Furthermore, this anti-inflammatory effect is characterized by a polyvalena in terms of efficacy in most pharmacological studies.

In contrast to the other experimental inflammation inhibitors, neither ulceration nor glycoproteins B is observed.

b) Stimulation of the non-specific defense mechanisms.

This stimulation is examined in the sample with "clearance w of coal in mice, based on the technique of Halpern, which consists in injecting into the ocular sinus a suspension of colloidal carbon on the animal and depending on the time the kinetics of it are evaluated. occurrence of the disappearance of the charcoal in the blood, taking measurements of the optical density.

The compounds are administered to the animal by intraperitoneal route 24 and 48 hours prior to the experiment, and the results are expressed as a percentage of activity compared to control animals which have received colloidal carbon injection alone: 146724 9

Substances Doses $ activity Control 8 min. after 30 min. after ___________ _ injection injection

Glyoproteins A 1 fflgAg 50 $ 81 $

Glycoproteins B 1 mgAg 52 $ 84 $

Glycoproteins A 0.5 mgAg 46 $ 77 $

Glycoprotein B 0.5 mgAg 45 $ 82 $

These results show the intense stimulation elicited by these two substances on the organism's defense mechanism pain.

c) Antibacterial action of the slow g and B-glycoproteins produced by the method of the invention.

The slow α and β glycoproteins trigger intense and lasting antibacterial protection in very small doses. This effect is polyvalent and occurs against both gram-positive and gram-negative germs. The effect is more preventive than curative.

The substances are administered to mice before the germ injection.

At a dose of 1 µg, the glycoproteins A and glycoproteins B provide the same percentage protection against Klebsiella pneumoniae, namely $ 90. At the same dose and for the same substances, protection is $ 30 against staphylococci. At a dose of 20 yAg, it is at $ 77 against the same microorganism.

The antibacterial effect on Klebsiella pneumoniae varies depending on the time of administration of the slow α and β glycoproteins, namely whether they are administered 48 hours before the infection, at the moment of infection or 48 hours after the infection. The protection is $ 100, $ 95 and $ 10 respectively. Thus, the slow α and β glycoproteins have a very clear preventive antibacterial effect.