JPS6363560B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel glycoprotein extracted from Klebsiella pneumoniae, a method for obtaining the same, its use as a drug, and a composition containing the same. Several French patent specifications have already described glycoproteins extracted from Klebsiella pneumoniae, for example French patent no.
No. 2043475, No. 2088112 and No. 2171907 are mentioned. These reported glycoproteins have molecular weights greater than ¹⁰⁶ daltons, contain approximately 62% bound hexoses and as much as 9% protein, and are highly susceptible to quaternary ammonium ions such as cetylpyridinium chloride or bromide. It is like being soluble. The present application is concerned with more precisely defined glycoproteins, the subject of which is novel water-soluble glycoproteins extracted from Klebsiella pneumoniae, which contain 10-20% protein and 50-70% protein. Neutral ose and 15 to 25 No. glucuronic acid and 1 to 2%
osamine and has a molecular weight of 80,000 to 350,000 Daltons. Neutral ose means in particular neutral hexoses, such as glucose, mannose or galactose. Preferably, the glycoprotein as defined above is characterized by its molecular weight estimated by ultracentrifugation of approximately 100,000 daltons. The glycoprotein of the present invention can be extracted from different strains of Klebsiella pneumoniae, but in particular from the strain deposited with the Patsur Institute under No. 52145. Structural studies of the above-mentioned glycoproteins using different chemical techniques, in particular reduction of uronic acid residues with carbodiimides, permethylation, uronic acid decomposition, periodic acid oxidation or oxidation with chromium oxide, have shown that the composition of the products according to the invention and This made it possible to specify the structure. Glycoproteins according to the invention are formed from protein chains onto which polysaccharide moieties are grafted. Preferred glycoproteins according to the invention are those in which the protein portion is composed of about 30% acidic amino acids. Acidic amino acids are amino acids such as aspartic acid or glutamic acid, i.e.
It is understood that it is an amino acid with a side chain having a COOH group. Preferably, glycoproteins according to the invention include those in which the N-terminal amino acid of the protein moiety is aspartic acid. Further, the preferred glycoprotein according to the present invention has a polysaccharide moiety of about 9.5 to 10.5 per molecule of galactose.
molecules of glucose, 4 to 5 molecules of mannose, and 3
~3.5 molecules of glucuronic acid. Among these, the polysaccharide part in particular has a structural formula consisting essentially of repeating tetrasaccharide units of. Among the novel glycoproteins which constitute the subject matter of the present invention, in particular the polysaccharide portion is formed from two polysaccharide chains, each of which has a link between a glucosamine molecule at one end of the polysaccharide chain and an asparagine molecule at the protein chain. N-
Includes those bound to protein moieties through glycosidic bonds. The subject of the present invention is also a method for obtaining the above-mentioned novel water-soluble glycoprotein, which method comprises first preparing a solution of the glycoprotein obtained by dialysis of a lytic extract from a culture of Klebsiella pneumoniae. 4
The resulting precipitate is isolated and then dissolved using an aqueous solution of sodium chloride, the resulting aqueous glycoprotein salt solution is treated with a low molecular weight alkanol, and the resulting precipitate is isolated and dissolved using aqueous sodium chloride. The new precipitate is redissolved in water, dialyzed,
It is then characterized by being freeze-dried. Different solutions of glycoproteins can be used at the start of the method, these solutions having a molecular weight equal to or greater than a predetermined molecular weight, which is constant for these membranes. It is obtained by dialysis of a lysate from a culture of Klebsiella pneumoniae through a membrane capable of retaining molecules. The membrane used is, for example, the product name Amicon.
Although the membranes are sold under the names XM50, PM30 and UM2, it is preferred to use membranes with a retention threshold of 100,000 Daltons, such as those sold by Amicon under the trade names XM100 or HIP100. Particularly suitable membranes are capable of retaining molecules with a molecular weight greater than 300,000 Daltons, which are advantageously available under the trade name Amicon and Romicon.
It consists of a membrane sold as XM300. Diafiltration allows molecules with a molecular weight greater than a given molecular weight to be selected in solution, as described above by membrane selection, depending on the desired retention threshold. It is clear to the person skilled in the art that it is quite possible to use other solutions with the same properties obtained by other means, for example by chromatography on hydrophilic polymeric gels. Prior to this selection operation, the lysate can very advantageously be freed of lipids and nucleic acids. Under particularly suitable conditions for carrying out the method according to the invention, the starting solution of glycoproteins is obtained under Letter of Addition No. 2171907 to French Patent No. 2043475.
It is obtained by the method described in the specification. In this Patent Supplement, the molecular weight of glycoproteins is estimated by gel exclusion techniques. The quaternary ammonium used to treat the starting solution of glycoproteins is, for example, cetylpyridylammonium chloride, but especially cetyltrimethylammonium bromide or setapron. After treatment with quaternary ammonium, the resulting precipitate can be separated from the supernatant by standard methods such as decantation or filtration, but is preferably separated by centrifugation. be. The precipitate is then advantageously treated with sodium chloride.
Re-dissolve using 0.2M solution. The resulting solution is then poured cold, i.e. at about +4
For example, methanol, ethanol, n
- working with low molecular weight alkanols such as propanol or isopropanol, preferably ethanol is used; The most interesting results are obtained using 6 volumes of ethanol per 1 volume of aqueous salt solution overnight at a temperature of +4°C. The precipitate is redissolved in water and dialyzed for purification. Dialysis is carried out using standard cells sealed with eg collodion or cellulose membranes. Particularly included are cells in which the membrane is made of regenerated cellulose, the average diameter of the pores is equal to about 24 Å, and retains substances with a molecular weight greater than a value in the range of 12 to 14,000 Daltons. As dialysis cells, mention may be made in particular of Visking tubes. This dialysis makes it possible to remove from the solution of the glycoprotein any low molecular weight impurities present, as well as traces of alkanol. Apparently, the glycoproteins of the invention are obtained in slightly lower purity without carrying out a dialysis step. In preferred conditions for doing this, the above method comprises:
The method is characterized in that the quaternary ammonium used is cetyltrimethylammonium bromide, the first precipitate is isolated by centrifugation, and the low molecular weight alkanol is ethanol. A subject of the invention is also the glycoprotein obtainable by the method of the invention. The products forming the subject of the present invention have extremely interesting pharmacological properties. They have, in particular, pronounced antibacterial and immunostimulatory properties and very good solubility resistance. These properties are shown in the experimental section below. These properties justify the use of the glycoprotein of the present invention as a drug. Therefore,
The subject of the invention is also the use of the above-mentioned glycoproteins as drugs. The drugs of the present invention include those characterized by being composed of the glycoprotein obtained by the method of the present invention. These drugs are used, for example, in humans and animals for the treatment or prevention of infectious diseases caused by bacteria or viruses, for the treatment of diseases caused by parasites and toxic infections, and for post-morbid and postoperative infections. used for treatment. Typical dosages may vary depending on the product used, the person being treated and the condition in question, but for humans 0.5 to 10 mg daily for oral administration, 2 to 10 mg daily for rectal administration and parenteral administration. The daily dose is 0.25-5 mg. Finally, the subject of the invention is a pharmaceutical composition containing the glycoprotein of the invention as a pharmaceutical ingredient. As a drug, the glycoprotein of the present invention can be used in the digestive system,
It can be administered parenterally or topically. The corresponding pharmaceutical compositions can be, for example, solid or liquid and can be presented in the pharmaceutical forms currently used in human medicine, such as pure or sugar-coated compressed tablets, gelatin capsules, granules, solutions, syrups, etc. , suppositories, injection preparations, ovules, creams, ointments, lotions, drops and eye drops, which are manufactured according to conventional methods. The active ingredients are combined with excipients commonly used in these pharmaceutical compositions, such as talc, gum arabic,
Can be mixed with lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting agents, dispersing or emulsifying agents and/or preservatives. can. Examples for carrying out the present invention will be shown below, but the present invention is not limited to these. Example 1 20 g of the product obtained in Example 1 of FR 2 171 907 were dissolved in softened water 2. Approximately 1.6 of 3% Cetabron was slowly added and the whole was stirred for 1 hour and then centrifuged at 10000 rpm for 15 minutes. The precipitate was taken up in 0.5 ml of 0.2M sodium chloride solution and then 3 ml of 95% ethanol was added over 15 minutes. After stirring for 1 hour, centrifugation was performed at 10,000 rpm for 15 minutes, the resulting supernatant was removed, and the precipitate was
of water and then in a Visking tube.
Dialyzed against softened water for 48 hours at 4°C. After completing this dialysis, the solution was freeze-dried. 8.16g of the desired glycoprotein was obtained. Example 2 20 g of the product obtained in Example 1 of FR 2 171 907 were dissolved in 1 part softened water. While stirring, 3% Cetabron 0.800 was added.
After stirring for 1 hour, it was centrifuged at 10,000 rpm for 15 minutes. The resulting precipitate was dissolved in 0.2M sodium chloride solution.
0.250 and added 1.5 liters of 95% ethanol under stirring. After stirring for 1 hour, it was centrifuged at 10,000 rpm for 15 minutes. The supernatant was removed, the precipitate taken up in water 0.500 and +4 in a Visking tube.
Dialyzed against softened water at ℃ for 48 hours. After this dialysis is completed, the solution is freeze-dried and
9.4g of the desired glycoprotein was obtained. Example 3 Compressed tablets were manufactured according to the following formulation. Glycoprotein obtained in Example 1: 5 mg Excipients required to obtain 1 compressed tablet: 100 mg (Excipient details: lactose, starch, talc, magnesium stearate). Example 4 An ointment was prepared according to the following formulation. Glycoprotein obtained in Example 1 200 mg Required amount of excipients 100 g Pharmacological study A Antibacterial activity of the glycoprotein obtained by the method of carrying out the present invention The glycoprotein of the present invention has the following properties: Very low doses provided potent and long-lasting antibacterial protection.
This activity was broad-spectrum, acting on both Gram-positive and Gram-negative bacteria. The activity was prophylactic rather than curative. The products of the invention were administered to mice prior to injection of bacteria. At a dose of 10 r/Kg, the "glycoproteins" of Examples 1 and 2 ensured a 100% prevention rate against infections equivalent to 500 LD20 of Klebsiella pneumoniae. For the same dose and same product, prophylaxis against Klebsiella pneumoniae
It was 85% for the infection equivalent to 12,500LD50. At a dose of 100 r/Kg, it was 100% vs. 12500 LD50 of this same microorganism. Antibacterial activity against Klebsiella pneumoniae varied depending on the timing of administration of the glycoproteins, with protection being 30% and 100%, respectively, depending on whether they were administered 48 or 96 hours before infection; The glycoprotein according to the invention therefore had a very clear antibacterial activity as a prophylactic. B. Stimulation of non-specific defenses This stimulation is the “removal” of carbon in mice.
The study was conducted using a technique perfected by Halpern, which involved injecting a suspension of colloidal carbon into the eye of an animal and measuring the rate of disappearance of carbon into the blood as a function of time. Therefore, the optical density is measured. The product was administered to the animals by the intraperitoneal route 24 and 48 hours before the test and the results were compared with the control case in which colloidal carbon alone was injected to determine the percent activity.
It was written as.

Table 1 Considering these results, it can be noted that these two products provide a strong stimulation of protective production. C Acute toxicity: lethal dose by intraperitoneal administration in mice 50
(LD50) was determined by the method of Behrens and Culver. For the glycoproteins of Examples 1 and 2, it was 100 mg/Kg. D Solubility Tolerance Example 1 at a dose of 1000r/Kg in mice
A subcutaneous injection of 0.2 ml of glycoproteins 2 and 2 did not cause any local or systemic intolerance.

Claims (1)

[Claims] 1. 10-20% protein and 50-70% neutral ose
Obtained by dialysis of a lytic extract of a culture of Klebsiella pneumoniae, characterized by containing 15-25% glucuronic acid and 1-2% osamine and having a molecular weight of 80,000-350,000 Daltons. A novel water-soluble glycoprotein produced by further processing glycoprotein. 2. The glycoprotein according to claim 1, which has a molecular weight of about 100,000 Daltons. 3. The glycoprotein according to claim 1 or 2, which is extracted from a bacterial strain deposited with the Patsur Research Institute as No. 52145. 4. The glycoprotein according to any one of claims 1 to 3, wherein the protein portion consists of about 30% acidic amino acids. 5 Claim 1, characterized in that the N-terminal amino acid of the protein portion is aspartic acid.
The glycoprotein according to any one of Items 1 to 4. 6 The polysaccharide moiety is approximately per molecule of galactose.
Claims 1 to 5, characterized in that it contains 9.5 to 10.5 molecules of glucose, 4 to 5 molecules of mannose, and 3 to 3.5 molecules of glucuronic acid.
The glycoprotein according to any of paragraphs. 7 Structural formula of polysaccharide part 7. Glycoprotein according to claim 6, characterized in that it consists essentially of repeating tetrasaccharide units of. 8 The polysaccharide moiety is formed from two polysaccharide chains,
Claims 1 to 7, each of which is bonded to the protein moiety by an N-glucoxide bond between a molecule of glucosamine at one end of the polysaccharide chain and a molecule of asparagine at the protein chain. The glycoprotein according to any one of. 9. A solution of glycoproteins obtained by dialysis of a lytic extract of a culture of Klebsiella pneumoniae is treated with quaternary ammonium, the resulting precipitate is isolated, and then treated with an aqueous solution of sodium chloride. The resulting aqueous glycoprotein salt solution is treated with a low molecular weight alkanol while cold, and the resulting new precipitate is redissolved in water.
characterized by dialysis followed by lyophilization,
10-20% protein and 50-70% neutral ose and 15-
A method for obtaining a water-soluble glycoprotein containing 25% glucuronic acid and 1 to 2% osamine and having a molecular weight of 80,000 to 350,000 daltons. 10. Claim 9, characterized in that the quaternary ammonium used is cetyltrimethylammonium bromide, the first precipitate is isolated by centrifugation, and the low molecular weight alkanol is ethanol. The method described in section. 11 Extracted from Klebsiella pneumoniae containing 10-20% protein, 50-70% neutral ose, 15-25% glucuronic acid and 1-2% osamine and having a molecular weight of 80,000-350,000 Daltons. An antibacterial or immunostimulant composition comprising at least one water-soluble glycoprotein as an active ingredient.