JPS59219236A - Method of obtaining slow active alpha and beta glycoprotein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Belgian patent No. 750.641 in the name of the applicant discloses that slow α-glycoproteins and β-glycoproteins are produced by extraction of a culture of a microbial strain that has undergone prior lysis. A method for obtaining a mixture with proteins is described.

The glycoproteins obtained by this method have very interesting therapeutic properties. They are endowed with considerable anti-inflammatory properties and rapid and non-specific immunity. Furthermore, they have the advantage that they do not cause allergies or high fever, and that they do not cause any untoward phenomena upon injection.

The structure of these slow α- and β-glycoproteins is demonstrated by the electrophoretic behavior under density gradients shown for known reference glycoproteins, and in particular for plasma glycoproteins. The slow α- and β-glycoproteins of the present invention are glycoproteins whose electrophoresis is very slow when compared to a known reference glycoprotein (orosomucoid) under certain electrophoretic conditions. , which exhibits the same electrophoretic behavior as plasma α- and β-glycoproteins. Four types of plasma glycoproteins, α, α2, β, and γ, have been separated, and their electrophoretic behavior is also known (Anal,
Chemistry 50 (195B), p12
6-127).

The chemical properties of these substances are determined by chemical reactions such as their hexose and pentose content; by approximate identification of their molecular weight using selective chromatographic reagents such as modified cellulose, Sephadex gel or molecular sieves; Protein (P
rotidic) fraction content; by hexosamine content; and by sialic acid content.

The mixture of slow-acting α- and β-glycoproteins obtained by the above method is composed of slow-acting thermostable α- and β-glycoproteins that are soluble in acids and soluble in ammonium sulfate solution. It can be defined as

The slow α- and β-glycoproteins obtained by this method also have a bound hexose content between 50 and 60%.

The present invention relates to Klebsiella pneu
Obtaining slow α- and β-glycoproteins in purified form from a known mixture of slow α- and β-glycoproteins produced by known methods from cultures of known strains of P. moniae) One or more of the known mixtures of slow α- and β-glycoproteins act as molecular sieves.
Purified by diafiltration of an aqueous solution through one or more membranes with defined apertures, separating the inactive or slightly active fraction, resulting in a very pure fraction retained by the membrane in a diafiltration cell. The purpose of the present invention is to provide a method for obtaining purified slow-acting α- and β-glycoproteins, which is characterized in that the two fractions are collected, chromatographed on a hydrophilic polymer gel, and the unfixed fractions are collected. do.

The method of the invention makes it possible to obtain slow α- and β-glycoproteins in very pure and therefore very active form. It also makes it possible to remove a large part of the protein degradation products that could have remained in the mixture, thus making it possible to obtain the final product in highly purified form.

The method of the invention is particularly characterized by the following points.

(1) The membrane used for diafiltration is a reticulated selectively permeable membrane, preferably with the trademark "Amic on".
XM! +00'' (manufactured by Mikon Co., Ltd., USA).

(2) Also, the selectively permeable membrane is preferably
AJnicon XM 50, PM30 and UM 2
It is commercially available as ``.

(31 Hydrophilic polymer gel is Sepharose Gel 6B.

(4) Moreover, the hydrophilic polymer gel is Sephadex gel q200.

The purified slow α- and β-glycoproteins obtained by the method of the present invention have a molecular weight of 772
Depending on the ratio of hexose content and r defined.

The very pure fraction obtained according to the method of the invention is 1
It consists of glycoproteins with a molecular weight equal to or greater than 0' daltons, containing between 60 and 65% hexoses, giving a monoalpha white ratio close to 7. Generally, it contains about 62% bound hexose. Nevertheless, due to the difficulty in standardizing the proportion of sugars, this content is 60-60%, since no conclusions can be drawn about the purity of both components.
It can vary between 5%.

The slow α- and β-glycoproteins obtained by the method of the present invention are useful as anti-inflammatory agents and stimulants for non-specific defense of living organisms. They are particularly useful in the treatment of bone and joint infections, bronchitis and bacterial infections.

They can be used as active ingredients in pharmaceutical compositions adapted for administration parenterally, lingually, orally, rectally, mucosally or topically.

Mention may be made, for example, of VC-prepared injectable solutions or suspensions in ampoules or multi-dose bottles, sublingual tablets, compressed tablets with enteric coating, aerosols, sprays, creams, ointments, lotions, herbal medicines or pills. .

Effective dosages vary depending to a large extent on therapeutic instructions and mode of administration.

The following examples illustrate the invention without limiting it.

According to the method described in Belgian Patent No. 750.641, the selected bacterial strain Klebsiellap
pneumoniae NCTC5055 (or Institut Pasteur Deposit No. 452145)) is cultured in a fermenter using an appropriate medium. When the culture ceases, the microorganisms are allowed to lyse for 8 days until the bacterial suspension becomes stable.

The lysate is then concentrated and lipids removed from it with a methylal-methanol mixture (4-1). The centrifugation residue is washed with methanol, dissolved in water and mixed with formol. The bacterial extract is finally processed by precipitation of the aqueous solution with a methylal-methanol mixture and washing of the precipitate with methanol.

(2) Properties of bacterial extract The extract obtained is clear and soluble in aqueous media.

5 in 0.6N perchloric acid, 10% (p/v) trichloroacetic acid medium or p) i = 7.0 and 5 at 15 °C.
．． No precipitation in 6M ammonium sulfate. Partial precipitation is caused by manganese sulfate or cetylpyridinium chloride (C,P
, C,).

The main components of this bacterial extract were evaluated, which specifically showed a bound neutral hexose content of 26-32% and an α-amino nitrogen of 6.2,× by batch method.

Klebsellia was prepared according to the above-mentioned preparation method.
2 g of entrained α- and β- obtained starting from a culture of P. pneumoniae N c'rc 5055).
Dissolve the protein mixture in 400 cc of water. This solution is placed in a dialysis cell closed with a membrane made of AXTIicon The dialysis is continued by constantly shifting the dialysis balance by adding an equal volume of distilled water for this purpose.This operation is done continuously and the total diafiltered volume is equal to 10 times the initial volume. The diafiltration operation is considered to have been completed when the dialysis cell reaches 50% or more.
6B, and the portions that were not fixed on Sepharose gel 6B were collected and lyophilized.

Also, for diafiltration, first use Ar11icon PM 30
in a dialysis cell containing a membrane made of ArFl.
It can be carried out several times in a second dialysis cell with a membrane made of Aln1con X50 and finally in a third diafiltration cell with a membrane made of Aln1con After chromatography on 6B and lyophilization of the rejected fractions, fractions identical to those obtained by a single diafiltration through the membrane are obtained.

Thus obtained (Am1con XM 30
0 membrane and is of the amount from Sepharose gel 6B. These macromolecular materials have a higher hexose composition than the materials obtained by the process of the Belgian patent mentioned above, ie, both the impure fraction. It is generally close to 6296. On the other hand, the α-aminochloride content is small (1,6X) and lower than in both cases of the storehouse obtained previously.

The mobile α- and β-glycoproteins obtained in this way are separated by a 8M urea solution or during reducing agent solution diafiltration, and the low molecular weight molecules spontaneously regenerate when the dissociating agent is removed. We can meet. Nevertheless, the newly obtained macromolecules are Arn1 con Xhl
Unlike the slow α- and β-glycoproteins retained on the 500 membranes, therefore several enzymes (pronase, trypsin, pancreatin, hyaluronidase) act on the slow α- and β-glycoproteins. It was determined that a small fraction of the macromolecule was sensitive to these enzymes and that its pharmacological activity did not appreciably change if the macromolecules were subsequently fractionated by a membrane. Ru.

The purification yield according to the above manufacturing method is about 30-35%.

The slow α- and β-glycoproteins thus obtained have a bound hequinose content of about 62%, a proteinaceous substance content close to 9%, and *, iZ:,'4 ；、、
The ratio of 2 is 11 to 7. However, this exact explanation will be difficult. This is because standardization by orcinol staining shows that the lagging α- and β-glycoproteins are actually contained in the form of galactose and mannose. This is because it is not done regarding the class.

Structural studies Acid hydrolysis under vacuum reveals different molecular components after separation on cellulose plates. Therefore, a very large variety of amino acids can be represented, which indicates the mobility (χ
- and β-glycoproteins from peptidoglycan and osamine.

Among the sugars the presence of glucose, galactose and mannose is established.

The presence of ribose was not shown.

After alkaline hydrolysis with cleavage of the 0-osidyl bond, the study of the fragments formed has shown that long glycans (
Glycanic) chains are grafted onto a ring of small chains of protein.

Therefore, they are glycoprotein V=
It has molecules of the same type. Furthermore, since it cannot be precipitated by cetylpyridinium chloride, there are no various types of solidification.

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

Pharmacological study 3) Anti-inflammatory activity Carrageenin was locally injected into the paws of rats to form plantar inflammatory edema. The test compound was administered intraperitoneally 1 hour before the stimulus injection. Paw volumes were then measured before and after stimulant injection. Since the increase in paw volume is a measure of the degree of inflammation, r) the A4o dose (ie the dose that preserves 40% of treated animals compared to control animals) was determined.

Comparisons were made between the substance (glycoprotein hole) produced as described in [(■) Preparation example of starting pongee fungi extract] and purified lagging a- and β produced by the method of the present invention. - Glycoprotein (glycoprotein B') and the most commonly used anti-inflammatory drugs.

The results are as follows.

It can be seen that the glycoprotein B of the present invention is the most effective anti-inflammatory agent among the tested compounds. Note that glycoproteins A and B had no ulcer-inducing properties compared to other compounds.

b) Stimulation of non-specific defenses This stimulus was studied by means of the "carbon purification test" in mice according to the method developed by Halpern. The method consists of injecting a suspension of colloidal carbon into the ocular side of the animal and evaluating the loss of carbon into the blood as a function of time by measuring optical density.

The test compound was administered intraperitoneally 24 hours and 48 hours before the test. Results are expressed as percentage removal of carbon particles compared to a control in which only colloidal carbon was injected and corresponded to 100X the number of carbon particles.

C) Mice were administered antibacterial active glycoprotein B and then injected with microorganisms. At a dose of 1 μEl 1 kg, glycoprotein B
90X of treated mice were protected from death.

Furthermore, against staphylococci, a dose of 20 μEl/kg protected 77X mice from death.

Claims (1)

[Claims] (1) Klebsiella Pneumo
In obtaining the slow α- and β-glycoproteins in purified form from a known mixture of slow α- and β-glycoproteins produced by known methods from cultures of known strains of P. niae), The known mixture of mobile α- and β-glycoproteins is purified by dialysis in an aqueous solution with one or more defined aperture membranes of one or more types acting as molecular sieves, The inactive or slightly active fraction is separated, the very pure fraction retained by the membrane is collected in a diafiltration cell, and the fraction is transferred onto a hydrophilic polymeric gel.
1. A method for obtaining purified slow-acting α- and β-glycoproteins, characterized in that the fractions that have not been fixed and fixed have been collected.