EA045761B1 - COMPOSITION OF OLIGOMERIC SUGAR DICIDS PRODUCED FROM ALGIN ACID - Google Patents

Description

Field of technology to which the invention relates

This invention relates to an optimal composition of oligomeric sugar diacids derived from alginic acid, created by selection for biological activity using an animal model of Alzheimer's disease to determine the effect of the degree of polymerization of oligosaccharides derived from alginic acid and their ratios on biological activity. As a result of the selection, a composition with the best biological activity was obtained, and the desired substance was isolated by membrane ultrafiltration.

Prerequisites for creating the invention

Alginic acid oligosaccharides have attracted much attention for their potential use for medical purposes. Alginic acid oligosaccharides are usually prepared from alginic acid used as raw materials in several steps.

In the molecules of alginic acid oligosaccharides from raw materials there are M regions formed by D-mannuronic acid molecules connected by p-1,4-glucosidic bonds; G regions, formed by L-guluronic acid molecules connected by α-1,4-glucosidic bonds, and MG regions, formed by hybridization of these two saccharides. Below are the structural formulas of Dmannuronic acid and L-guluronic acid in the form of formula (I) and formula (II).

The structure of the oligosaccharide obtained from alginic acid is shown below by formula (III)

Fragments M and G can be isolated from natural raw materials - alginic acid.

A conventional method can be summarized below as follows: alginic acid is first cleaved so that a mixture of the polysaccharides polymannuronic acid and polyguluronic acid is formed, then this mixture is subjected to acid precipitation so that some of the polyguluronic acid is removed from it (See, for example, methods described in Chinese Patent Application No. 98806637.8 and CN 02823707.2).

One method for preparing oligomeric mannuronic acid is as follows: the intermediate fragment M obtained as described above is subjected to further acidolysis by heating in an acidic environment, resulting in small fragments of the mannuronic acid polymer with the desired molecular weight. In addition, the cleavage efficiency can be improved by oxidative cleavage, in particular, the reducing end can be oxidized to form an open ring structure of a dicarboxylic sugar acid (for details, see Chinese Patent Application No. 200580009396.5 (No. 1 in the patent reference list) , filed by Meiyu Geng, et al., and US Patent No. 8835403 B2 (No. 2 in the patent list)). For convenience of description, further references to Patent Literature Nos. 1 and 2 are referred to collectively as prior documents, which are incorporated herein by reference in their entirety.

The reaction for producing mannuronic diacid described in the previous documents can be represented by reaction equation (V), namely, the aldehyde group at the C1 position of the mannuronic acid residue at the reducing end of the oligomannuronic acid polysaccharide is oxidized to form a carboxyl group.

In the above-mentioned oxidative conversion process, an alkaline solution of copper sulfate (Fehling's reagent) is usually used as an oxidizing agent. Previous documents describe the use of this oxidation method. More specifically, the following is done: in an alkaline environment, the reaction substrate - polymannuronic acid (that is, the intermediate product mentioned above - fragment M) is added to a solution of copper sulfate and kept in a boiling water bath for a period of time lasting from 15 minutes to 2 hours. In this method, the Cu ²⁺ ion oxidizes the aldehyde group, resulting in the formation of copper oxide, which precipitates as a brick-red precipitate. This reaction is often used to determine reducing sugars.

Previous documents have described that oligomannaric acids have activity against Alzheimer's disease (AD) and diabetes mellitus, with mannaric acid oligomers with a degree of polymerization of 6 having the greatest activity. The pathogenesis of Alzheimer's disease and type II diabetes mellitus is closely related to amyloid proteins ( β-amyloid and amylin). Amyloid proteins aggregate to form protein oligomers, which then aggregate to form fibers. These protein aggregates are cytotoxic and cause oxidative reactions in cells, leading to mitochondrial damage; and initiate a cascade of reactions such as inflammatory reactions, ultimately damaging many neurons and β cells, so that Alzheimer's disease and type II diabetes eventually develop. Oligomannaric acids target amyloid proteins, thereby counteracting the cascade of reactions initiated by amyloid proteins, thereby preventing/treating Alzheimer's disease and type II diabetes.

In order to obtain oligomannaric acid having the activity against Alzheimer's disease and type II diabetes described in previous documents, it is necessary to isolate guluronic acid from the alginic acid starting material. Alginic acid usually contains more than 30% guluronic acid, up to a maximum of about 70%. Thus, the actual costs associated with obtaining high-purity oligomannaric acid are very high.

Disclosure of the Invention

In a first aspect, this invention relates to a composition of oligomeric sugar diacids derived from alginic acid, comprising mannuronic acid described by formula (IV) and/or guluronic acid, or pharmaceutically acceptable salts thereof

Np ^soon

BUT Formula (IV) where n is an integer selected from numbers from 1 to 9, m is selected from 0, 1 or 2, m' is selected from 0 or 1, and where the total mass of oligomeric sugar diacids obtained from alginic acid with n= 1-5 is no less than 60% of the total weight of the composition, and the total weight of guluronic diacids is no more than 50% of the total weight of the composition.

In another aspect, the present invention relates to a pharmaceutical composition or medical product that contains the above composition of alginic acid-derived oligomeric sugar acids. In other aspects, the present invention also relates to the use of said composition of alginic acid-derived oligomeric sugar acids in the treatment of diseases selected from Alzheimer's disease, Parkinson's disease, inflammatory diseases, pain, diabetes mellitus or vascular dementia.

In particular, the composition of oligomeric sugar acids obtained from alginic acid according to this invention is a mixture of mannuronic and guluronic acids with different degrees of polymerization, and the main components are oligosaccharides with a degree of polymerization from 2 to 10, namely: the M region of alginic acid formed by the residues mannuronic acid connected by β-1,4-glycosidic bonds; alginic acid G region, formed by guluronic acid residues connected by a-1,4-glycosidic bonds; alginic acid MG region formed by hybridization of these two saccharides. Mannuronic diacids are known to have some pharmacological activity against Alzheimer's disease and diabetes mellitus. The most active saccharides in this regard are penta-, hexa-, hepta- and octasaccharides, especially hexasaccharides. However, the present inventors have discovered that a mixture of mannuronic and guluronic acids with degrees of polymerization from 2 to 10 also has pharmacological activity against Alzheimer's disease and diabetes mellitus, and it is important to control that the content of guluronic acids is within a certain range. In other words, the composition of oligomeric sugar diacids obtained from alginic acid according to the invention can be obtained at significantly lower costs, which facilitates its practical production, including on an industrial scale.

Brief description of drawings

Fig. 1 - NMR spectrum of the intermediate product.

Fig. 2 - mass spectra of disaccharides, trisaccharides and tetrasaccharides in product A.

Fig. 3 - mass spectra of pentasaccharides, hexasaccharides and heptasaccharides in product A.

Fig. 4 - mass spectra of octasaccharides, nonasaccharides and decasaccharides in product A.

Fig. 5 - NMR spectrum of product A.

Fig. 6 - NMR spectrum of product B.

Fig. 7 - NMR spectrum of product C.

Fig. 8 - NMR spectrum of product D.

Fig. 9 demonstrates the effect of different compositions of oligosaccharides and mannuronic diacid hexamer on the number of platform crossings in rats with Alzheimer's disease. The values along the abscissa axis in this figure are as follows: i - control group; ii - model group; iii product A; iv - product B; v - product C; vi - product D; vii - hexamer of mannuronic diacid.

Fig. 10 shows the effect of various compositions of oligosaccharides and mannuronic diacid hexamer on the swimming distance of rats with Alzheimer's disease. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 11 demonstrates the effect of various compositions of oligosaccharides and mannuronic diacid hexamer on the duration of dismounting from the rod in rats with Parkinson's disease on day 11. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 12 shows the effect of various compositions of oligosaccharides and mannuronic diacid hexamer on the duration of latency in rats with Parkinson's disease on day 11. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 13a, 13b demonstrate the therapeutic effect of various compositions of oligosaccharides and mannuronic diacid hexamer on inflammatory enteritis in mice. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 14 demonstrates the effect of various compositions of oligosaccharides and mannuronic diacid hexamer on postprandial blood glucose levels in diabetic mice. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 15 demonstrates the effect of various compositions of oligosaccharides and mannuronic diacid hexamer on the latency of writhing in acetic acid-treated mice. The designations along the abscissa axis in this figure are as follows: i - model group; ii - product A; iii - product B; iv - product C; v - product D; vi is a hexamer of mannuronic diacid.

Fig. 16 shows the effect of various compositions of oligosaccharides and mannuronic diacid hexamer on the number of writhings in mice administered acetic acid. The abscissa axis symbols here are the same as in Fig. 15.

Fig. 17 demonstrates the effect of different compositions of oligosaccharides and mannuronic diacid hexamer on the amount of head rubbing in rats with nitroglycerin-induced migraine. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 18 demonstrates the effect of different compositions of oligosaccharides and mannuronic diacid hexamer on the number of c-fos-expressing cells in the caudal spinal trigeminal nucleus in rats with migraine induced by electrical stimulation of the trigeminal ganglion. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 19 demonstrates the effects of different compositions of oligosaccharides and mannuronic diacid hexamer on dark avoidance latency in mice with vascular dementia caused by bilateral carotid artery occlusion. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 20 demonstrates the effect of different compositions of oligosaccharides and mannuronic diacid hexamer on the number of errors in the dark avoidance test in mice with vascular dementia caused by bilateral carotid occlusion. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 21 demonstrates the effects of different compositions of oligosaccharides and mannuronic diacid hexamer on water maze escape latency in mice with vascular dementia caused by bilateral carotid artery occlusion. The abscissa axis symbols here are the same as in Fig. 9.

Fig. 22 demonstrates the effect of different compositions of oligosaccharides and mannuronic diacid hexamer on the number of platform crossings in mice with vascular dementia caused by bilateral carotid artery occlusion. The abscissa axis symbols here are the same as in Fig. 9.

Detailed Disclosure of the Invention

Various aspects of the present invention are described in detail below, but the invention is not limited to these specific embodiments. Those skilled in the art will be able to make certain changes and additions substantially consistent with the description below, and these changes and additions are also included within the scope of the present invention.

Composition of oligomeric sugar diacids obtained from alginic acid

In a first aspect, this invention relates to a composition of oligomeric sugar diacids derived from alginic acid, containing mannuronic acid described by formula (IV) and/or guluronic acid, or their pharmaceutically acceptable salts ^coon

Formula (IV) where n is an integer selected from 1 to 9, m is selected from 0, 1 or 2, m' is selected from 0 or 1, and where is the total mass of oligomeric sugar diacids derived from alginic acid with n=1 -5

- 3 045761 is no less than 60% of the total weight of the composition, and the total weight of guluronic diacids is no more than 50% of the total weight of the composition.

The composition of oligomeric sugar acids obtained from alginic acid according to this invention is a mixture of mannuronic and guluronic acids with different degrees of polymerization; the main components of this composition are oligosaccharides with degrees of polymerization from 2 to 10, namely the M region of alginic acid, formed by mannuronic acid residues connected by β-1,4-glycosidic bonds; alginic acid G region, formed by guluronic acid residues connected by a-1,4-glycosidic bonds; alginic acid MG region formed by hybridization of these two saccharides. According to previous applications, it is known that mannuronic diacids have pharmacological activity against Alzheimer's disease and diabetes mellitus, the most active saccharides in this regard being penta-, hexa-, hepta- and octasaccharides, especially hexasaccharides. Contrary to what was previously known, the inventors discovered that a mixture of oligomers of mannuronic and guluronic acid with degrees of polymerization from 2 to 10 also has pharmacological activity against Alzheimer's disease and diabetes mellitus, but should be controlled to ensure that the content of guluronic acids is in a certain range.

In practice, after preliminary cleavage of the original alginic acid as described above, the guluronic acid content of the resulting product is usually more than 30%, at most about 70%. If one follows what has been described in previous applications to obtain oligomannaric acid with high activity, it is necessary to remove guluronic acid to the maximum extent possible. However, based on the discovery made by the inventors mentioned above, there is no need to isolate and remove guluronic acid from the alginic acid degradation products. In addition, the inventors have discovered that if the content of guluronic acid is controlled within a certain range by adjusting the acid precipitation conditions, it is possible to achieve the same or even higher activity of the composition compared to the mannaric acid hexamer described in previous applications. In addition, since it is not necessary to remove guluronic acid as an impurity, the yield of the product is significantly higher than the yield of the product described in previous applications. Thus, the cost of the process of obtaining the desired product is significantly reduced, waste is reduced, and thereby practical production, including on an industrial scale, is facilitated.

According to one preferred embodiment of the proposed composition of oligomeric sugar diacids derived from alginic acid, the total mass of oligomeric sugar diacids derived from alginic acid, in which n=1-5, is 80-95% of the total mass of the composition, and the total mass of guluronic acid constitutes no more than 50% of the total weight of the composition.

According to one of the preferred embodiments in the proposed composition of oligomeric sugar diacids obtained from alginic acid, the ratio of the total mass of oligomeric sugar diacids with low degrees of polymerization, in which n = 1-3, to the total mass of oligomeric sugar diacids with low degrees of polymerization, in which n =4-7, ranges from 1.0 to 3.5.

According to one of the preferred embodiments in the proposed composition of oligomeric sugar diacids obtained from alginic acid, the total mass of oligomeric sugar diacids obtained from alginic acid, in which m+m'=1 or 2, is at least 50% of the total weight of the composition, preferably 60 %-90%, more preferably 70%-90%. In particular, in the composition of oligomeric sugar diacids obtained from alginic acid according to the invention, the total mass of oligomeric sugar diacids in which m+m'=1 is at least 10% of the total mass of the composition, preferably 30-40%. In another preferred embodiment, in the proposed composition of oligomeric sugar diacids obtained from alginic acid, the total mass of oligomeric sugar diacids in which m+m'=2 is at least 10% of the total mass of the composition, preferably 30-50%.

According to one preferred embodiment of the present composition of oligomeric sugar diacids derived from alginic acid, the total weight of oligomeric sugar diacids derived from alginic acid, in which n=1-5, is 80-95% of the total weight of the composition.

According to one preferred embodiment of the present composition of oligomeric sugar diacids derived from alginic acid, the total weight of oligomeric sugar diacids derived from alginic acid, in which n=1-3, is 20-70% of the total weight of the composition.

According to one of the preferred embodiments in the proposed composition of oligomeric sugar diacids obtained from alginic acid, the ratio of the total mass of oligomeric sugar diacids, in which n = 1-3, to the total mass of oligomeric sugar diacids, in which n = 47, is from 1.0 to 3.5, preferably from 1.0 to 3.0.

According to one preferred embodiment of the proposed composition of oligomeric sugar diacids derived from alginic acid, the weight percentage of oligomeric sugar diacids derived from alginic acid with different degrees of polymerization is disaccharides 5-25%, trisaccharides 15-30%, tetrasaccharides 15-28%, pentasaccharides 10-25%, hexasaccharides 5-15%, heptasaccharides 3-10%, octasaccharides 2-5%, nonasaccharides 1-5%, decasaccharides 1-5%. In particular, in the composition according to the invention, the weight percentage of oligosaccharides is as follows:

disaccharides 10-20%, trisaccharides 18-30%, tetrasaccharides 15-28%, pentasaccharides 15-20%, hexasaccharides

5-10%, heptaxaccharides 3-5%, octasaccharides 2-3%, nonasaccharides 1-3%, decasaccharides 1-3%.

According to one preferred embodiment of the present composition of oligomeric sugar diacids derived from alginic acid, the total weight of guluronic acid, in which n=1-3, is 0.1-50% of the total weight of the composition, preferably 1-30%.

In the composition of oligomeric sugar diacids obtained from alginic acid according to the invention, the pharmaceutically acceptable salts of the oligomeric sugar diacids obtained from alginic acid are sodium or potassium salts.

Method for producing a composition of oligomeric sugar diacids from alginic acid

The method for producing oligomeric sugar diacids from alginic acid is briefly as follows.

As a result of the preliminary cleavage of alginic acid, a mixture of polysaccharides is obtained - polymannuronic and polyguluronic acids. This polysaccharide mixture is subjected to acid precipitation to remove some of the polyguluronic acid. In such acid precipitation, the higher the pH, the higher the content of polyguluronic acid in the resulting polysaccharide mixture (see, for example, the methods described in Chinese Patent Application No. 98806637.8 and CN02823707.2). In the presence of an oxidizing agent, chains of sugar residues in the polysaccharide molecules of the above mixture undergo oxidative cleavage, so that oxidized oligosaccharides with different degrees of polymerization are formed. In oxidized oligosaccharides, the mannuronic and guluronic acid residues at the reducing end of the oligosaccharide molecule are oxidized to sugar diacids containing 3-6 carbon atoms.

According to the invention, ozone is particularly preferred as an oxidizing agent for the oxidative decomposition reaction described above. In this process, oxidative cleavage of chains of sugar residues occurs when ozone is introduced into a solution containing a mixture of polysaccharides. Oxidative cleavage is carried out at a temperature preferably 0-70°C, more preferably 10-45°C. During the oxidative digestion step, the pH of the reaction mixture is 3-13, preferably 4-10, more preferably 6-8.

Processes previously used in the art such as oxidative decomposition using ozone, acid hydrolysis in the presence of an alkaline solution of copper sulfate (see the previous document) or hydrogen peroxide and sodium hypochlorite (see Chinese Patent Application No. 01107952.5) are similar in that that in all three methods, the cleavage of chains of sugar residues is achieved. The difference lies in the fact that the structure of the reducing end of the polysaccharide chain of the cleavage product is different. According to the invention, the reducing end of the oxidative cleavage product - mannuronic and guluronic acids - contains a dicarboxylic acid structure containing 3-6 carbon atoms. In addition, the method using oxidative cleavage according to the invention has other advantages: 1) the reaction conditions are moderate, no special conditions are required; 2) ozone for oxidation can be obtained at the reaction site, which reduces the difficulties associated with transportation in industrial production; 3) upon completion of the oxidation reaction, ozone spontaneously turns into oxygen without residual reagents harmful to the environment. The reaction process of the invention is described by equation (VI)

.CQW simplified image of the first reaction

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(VI)

In the above illustration of reaction (VI) and in the general formula (IV), the oligosaccharides in which m=2 and m'=1 are sugar acids with six carbon atoms at the end;

oligosaccharides in which m=1 and m'=1 or m=2 and m-0 are sugar acids with five carbon atoms at the end;

oligosaccharides in which m=1 and m-0 or m=0 and m-1 are sugar acids with four carbon atoms at the end;

oligosaccharides in which m=0 and m'=0 are sugar acids with three carbon atoms at the end.

In the composition according to the invention, the total mass of oligomeric sugar diacids obtained from alginic acid, in which n=1-5, is 80-95% of the total mass of the composition; the total mass of oligomeric sugar diacids obtained from alginic acid, in which n = 1-3, is 2070% of the total mass of the composition, and the ratio of the total mass of oligomeric sugar diacids, in which n = 1-3, to the total mass of oligomeric diacids, in which n=4-7, ranges from 1.0 to 3.5; preferably from 1.0 to 3.0. The total weight of guluronic acid is no more than 50% of the total weight of the composition, preferably from 0.1 to 50%, more preferably from 1 to 30%.

In an illustrative embodiment of the present invention, the production method includes the following steps:

(1) Preparation of oligomeric sugar diacids from alginic acid: Preparation of mixed polysaccharides of polymannuronic and polyguluronic acids. As mentioned above, according to this invention, mixed polysaccharides of polymannuronic and polyguluronic acids are used as feedstock; this material can be obtained by methods known in the art, for example the methods described in Chinese Patent Application No. 98806637.8 and CN 02823707.2. The commonly used method can be simplified as follows: alginic acid is first cleaved so that mixed polysaccharides, polymannuronic acid and polyguluronic acid, are formed. After the mixed polysaccharides are subjected to acid precipitation, the polyguluronic acid content is adjusted to obtain the desired mixture of polysaccharides containing polymannuronic acid and polyguluronic acid.

Oxidative splitting by ozone

The mixed polysaccharides are dissolved in an appropriate amount of water and stirred at room temperature or with heating. A continuous stream of ozone is introduced into the reaction mixture, thereby starting the reaction. The reaction mixture is adjusted to pH 3-13, preferably 4-10, more preferably 6-8, by adding dropwise dilute hydrochloric acid or dilute NaOH solution. The temperature is preferably maintained in the range of 0-70°C, more preferably 10-45°C. Upon completion of the reaction, the ozone supply is stopped and the pH is adjusted to neutral.

Membrane separation and purification

The product obtained as described above is transferred into a solution with a concentration of about 10%, this solution is passed through an ultrafiltration membrane to remove breakdown products of lower molecular weight than monosaccharides. The retentate is collected. Membranes with a molecular weight cutoff of 1000-3000 Da are used, preferably 2000 Da. The collected material is concentrated on a rotary evaporator and dried in vacuum, resulting in a mixture of oligomeric sugar diacids. Analysis of the products obtained in this way showed that they are mixtures of oligosaccharides with degrees of polymerization from 2 to 10, the content of which varies within certain proportions. This method is described in examples 1-3.

(2) Comparison of oligosaccharide compositions by activity

The pharmacological activity of the oligosaccharide composition of this invention was compared with the activity of oligomeric (hexamer) mannaric acid according to the previous application. The results of this comparison show that the pharmacological activity of the oligosaccharide composition of this invention is significantly higher than the activity of hexamannaric acid of the previous application. Without being bound by any theory, the authors believe that the greatest pharmacological activity of the composition is achieved when the weight ratio of disaccharides to hexasaccharides exceeds 60% and the total weight of guluronic acid is no more than 50% of the total weight of the composition. If the amount of guluronic acid exceeds 60%, then the activity decreases.

The present invention also provides a drug or medical product containing an oligosaccharide composition derived from alginic acid as described above and, if necessary, a pharmaceutically acceptable carrier or excipient.

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According to Remington's Pharmaceutical Sciences, Martin, E.W., ed., Mack Publishing Company, 19th edition (1995), methods for preparing medicinal products based on oligosaccharide compositions with varying levels of active ingredients are known or obvious to those skilled in the art based on the disclosure herein. inventions. Methods for producing pharmaceutical compositions according to the invention involve the inclusion in their composition of pharmaceutical excipients, carriers, diluents, etc.

The pharmaceutical preparations of the invention are prepared by methods known in the art, including mixing, dissolution or lyophilization methods.

The pharmaceutical composition of this invention is administered to patients by various routes suitable for the chosen route of administration, for example orally or parenterally (intramuscular, intravenous, topical or subcutaneous).

Thus, the composition of the present invention combining a drug with a pharmaceutically acceptable carrier (eg, an inert diluent or edible carrier) can be administered systemically, eg, orally. The drug substance and the pharmaceutically acceptable carrier may be enclosed in hard or soft gelatin capsules or compressed into tablets. For oral administration for therapeutic purposes, the active substance of the invention is combined with one or more excipients and administered in the form of tablets (swallowable, dissolved), lozenges, capsules, elixirs, suspensions, syrups, cylindrical tablets, etc. Such compositions and preparations must contain at least 0.1% active substance. However, the relative amount of active substance in a given unit dosage form of such compositions and preparations may, of course, vary and may range from about 1 to about 99 wt.% of a given unit dosage form. In compositions used for therapeutic purposes, the amount of active substance must be such that an effective dose is achieved.

Tablets, lozenges, pills, capsules, etc. may also contain binding agents (eg gum tragacanth, gum acacia, corn starch or gelatin); excipients (eg dicalcium phosphate); disintegrating agents (for example, corn starch, potato starch, alginic acid, etc.); glidants (eg magnesium stearate); sweeteners (eg, sucrose, fructose, lactose or aspartame) or flavoring agents (eg, peppermint, wintergreen essential oil, or cherry flavor). When the unit dosage form is capsules, in addition to these conventional materials, the preparation according to the invention may also contain a liquid carrier, for example vegetable oil or polyethylene glycol. Various other materials may also be present in the unit dosage form to serve as a coating or to otherwise modify the physical form of the drug. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar and the like. Syrups and elixirs may contain substances such as sucrose and fructose as sweeteners, methylparaben or propylparaben as preservatives, colors and flavoring agents (for example, cherry or orange flavoring). Of course, any material used for the manufacture of any unit dosage form must be pharmaceutically acceptable and non-toxic in the quantities in which it is used in that particular case. In addition, the active substance according to the invention can be included in a preparation or system providing its sustained release.

The active substance of the invention can also be administered intravenously or intraperitoneally by infusion or injection. To do this, prepare an aqueous solution of the active substance or its salt, if necessary together with a compatible and water-soluble non-toxic surfactant. You can also use dispersions in glycerin, liquid polyethylene glycol, triacetin and their mixtures with oils. Under normal conditions of storage and use, these drugs must contain preservatives to prevent the growth of microorganisms.

Dosage forms of the pharmaceutical preparations of the invention suitable for injection or infusion include sterile aqueous solutions or dispersions, or sterile powders of the active ingredients (if necessary enclosed in liposomes), including preparations for immediate use, suitable for the preparation of solutions or dispersions, administered by injection or infusion. In any case, the final dosage form must be sterile, liquid and stable under the conditions of manufacture and storage. The liquid carrier may be solvents or liquid dispersion media, including, for example, water, ethyl alcohol, polyhydric alcohols (for example, glycerin, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides, and suitable mixtures of these materials . Proper fluidity of the drug is ensured, for example, by the formation of liposomes, a certain particle size (in the case of dispersions) or the use of surfactants. To prevent the proliferation of microorganisms, various antibacterial and fungicidal agents are used (for example, parabens, chlorobutyl alcohol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include in the composition of this invention agents that provide the desired osmotic pressure,

- 7 045761 for example sugars, buffering agents or sodium chloride. For compositions administered by injection, absorption into tissues is prolonged by the use of absorption retarding agents (eg, aluminum monostearate or gelatin).

Sterile injection solutions are prepared by combining the required quantity of the active substance in a suitable diluent with various other ingredients listed above, depending on the requirements, and then sterilized by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, methods such as vacuum drying and lyophilization are preferred, which produce the active ingredient in powder form along with the desired ingredients present in the pre-filtered sterile solution.

Solid carriers that can be used in the compositions of this invention include fine solids such as talc, clay, microcrystalline cellulose, silicon, aluminum, and the like. The compositions of this invention may use liquid carriers such as water, ethyl alcohol or ethylene glycol, or a mixture of water, alcohol and ethylene glycol; the components of the composition are dissolved or dispersed in these vehicles in effective quantities, if necessary with the help of non-toxic surfactants. To achieve optimal properties for this application of the drug, auxiliary ingredients (for example, flavoring agents) and additional antimicrobial agents are added to it.

Thickeners (for example, synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified cellulose or modified inorganic materials) are also used to produce plastic pastes, gels, ointments, soaps and other products applied directly to the skin.

The amount of a substance or a mixture containing it necessary to create a therapeutic effect depends not only on the substance itself, but also on the method of administration of the drug, the nature and specificity of the disease to be treated, as well as on the age and condition of the patient, and ultimately on decisions of a doctor or other medical professional.

The above preparations can be presented in a single dosage form, that is, a combination of physically separate units forming a single dose of the active substance and suitable for administration to humans and other mammals. A single dosage form may be a capsule or tablet, or a set of capsules or tablets. Depending on the nature of the treatment, the amount of the active ingredient of this invention in a single dose of the drug varies or is selected from about 0.1 mg to about 1000 mg or more.

In another aspect of the present invention there is provided a pharmaceutical composition or medicinal product comprising an alginic acid oligosaccharide composition of the present invention and, if necessary, a suitable carrier.

Another aspect of the present invention provides the use of an alginic acid oligosaccharide composition for the treatment of Alzheimer's disease.

In another aspect of the present invention, there is provided a method of treating individuals suffering from Alzheimer's disease which comprises administering to the patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

Another aspect of the present invention provides the use of an alginic acid oligosaccharide composition for the treatment of Parkinson's disease.

In another aspect of the present invention, there is provided a method of treating individuals suffering from Parkinson's disease, which comprises administering to the patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

Another aspect of the present invention provides the use of an alginic acid oligosaccharide composition for the treatment of inflammation.

In another aspect of the present invention, there is provided a method of treating individuals with an inflammatory process, comprising administering to the patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

Another aspect of the present invention provides the use of an alginic acid oligosaccharide composition for the management of pain.

In another aspect of the present invention, there is provided a method of treating individuals suffering from pain, which comprises administering to the patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

Another aspect of the present invention provides the use of an alginic acid oligosaccharide composition for the treatment of diabetes mellitus.

In another aspect of the present invention, there is provided a method of treating individuals suffering from diabetes mellitus, which comprises administering to the patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

Another aspect of the present invention provides the use of an alginic acid oligosaccharide composition for the treatment of vascular dementia.

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In another aspect of the present invention, there is provided a method of treating individuals suffering from vascular dementia, which comprises administering to the patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

Pain in the context of this invention includes various pains, including acute pain; chronic pain; neuropathic pain; postoperative pain; chronic pain in the lower back; cluster headache; postherpetic neuralgia; phantom limb pain; pain caused by damage to the central nervous system; toothache; pain unresponsive to opioids; visceral pain; pain associated with surgery; pain from bone injuries: exhaustion and pain during childbirth; pain caused by burns (including sunburn); postpartum pain; migraine; angina pain; pain associated with the condition of the genitourinary tract (including pain due to cystitis); pain caused by vascular disorders; trigeminal neuralgia; intercostal neuralgia; pain during surgical incisions; pain due to chronic fasciitis; pain in the heel area; muscle pain; bone pain; joint pain; cancer pain; pain due to benign tumors, etc.

Inflammation in the context of this invention includes various inflammatory processes, including (but not limited to those listed here) acute inflammation, chronic inflammation, vascular inflammation, inflammation of the nervous tissue, inflammatory processes in the central nervous system (for example, multiple sclerosis, encephalomyelitis, etc.), inflammatory processes in the peripheral nervous system, arthritis (for example, osteoarthritis, sacroiliitis, psoriatic arthritis, rheumatoid arthritis, etc.), ankylosing spondylitis, inflammatory bowel diseases (for example, Crohn's disease, inflammation of the colon in ulcerative colitis), inflammation in diabetic trophic ulcers , systemic lupus erythematosus, inflammatory skin diseases (for example, psoriasis, atopic dermatitis, eczema), etc.

The alginic acid oligosaccharide compositions of this invention are prepared in a manner that differs from those previously known in the art in that it is not necessary to separate the M and G moieties of alginic acid. This greatly simplifies the production process and reduces its cost. The method for producing oligosaccharides from alginic acid according to this invention is characterized by simplicity of the reaction, high content of active ingredients in the product and the absence of residues of the original reagents. Through practical testing, it has been proven that the alginic acid oligosaccharide composition of this invention can prevent and treat Alzheimer's disease, diabetes mellitus, Parkinson's disease, various inflammatory processes, pain and vascular dementia.

Animal models and steps for determining pharmacodynamic activity

1. Animal model to determine the pharmacodynamics of the resistance of the studied drugs to Alzheimer's disease.

Alzheimer's disease was induced in rats by unilateral intraventricular injection of β-amyloid. The Morris water maze was used to assess learning and memory in experimental animals.

For these experiments, male Wistar rats with a body weight of 180-220 g were taken. The animals were randomly divided into groups of 14 individuals each: a control group with pseudo-operation (invasive measures without biological effects), a model group, and a group receiving an agent. First, the animals were anesthetized (intraperitoneal injection of sodium phenobarbital at a dose of 40 mg per 1 kg of body weight), then fixed in a stereotaxic apparatus. The skin was prepared and disinfected using conventional means, an incision was made to expose the vertex, and the location of the CA1 region of the hippocampus was determined as 3 mm posterior to bregma at a distance of 2.2 mm from the midline suture of the skull and 2.8 mm deep from the dura according to Rat Brain Stereotactic Atlas (BAO Xinming, SHU Siyun, Beijing, People's Medical Press, 1991, 28). In each of the model and agent groups, a microinjector was used to insert a needle vertically to the surface of the skull into the CA1 region of the right hippocampus. Animals were slowly (1 μl/min) injected with 5 μl of a concentrated β-amyloid preparation (Ab1-40 solution was prepared in phosphate buffered saline (PBS) with a concentration of 1.4 mg/ml and kept in an incubator at a temperature of 37°C within 5 days for aggregates to form). Upon completion of the injection, the needle was not removed for 5 minutes so that the amyloid was completely dispersed into the tissue; the needle was then slowly withdrawn. The incision was stitched and the rats were warmed until they woke up. In the control group, animals were injected with 5 μl of sterilized PBS, and everything else was done as described above. The therapeutic agent was administered 7 days before the intervention and continued to be administered until the end of the experiment.

The Morris water maze test was performed on the 11th day after the intervention.

Navigation test. Rats from all groups were trained to find a hidden underwater platform in a swimming pool for 5 consecutive days. We recorded how long it took each individual to reach it (the search period, or the latent period of rescue). If after approximately 90 s the animal did not find the platform, it was directed in the water towards it along a straight path and allowed to stand there for 30 s so that learning and memorization could occur.

Spatial search test

One day after completion of the navigation test, the platform was removed. Now each rat was put into the water at the starting point and recorded how many times it crossed the place where the platform was; We calculated the ratio (in percentage) of the distance that the animal swam in the quadrant where the platform was located to the total distance covered by it in the entire pool. Based on these data, the learning ability and memory of this individual were assessed.

2. Animal model to determine the pharmacodynamics of the antidote of the studied drugs to Parkinson's disease.

These experiments were carried out on mice. The animals were randomly divided into 8 groups of 14 animals each: control group; a model group in which mice received (MPTP) and a group receiving a therapeutic agent. Dividing into groups and administering drugs was carried out on the same day. In the control group and in the model group receiving MPTP, the animals were administered intragastric saline, and in the remaining groups, mice received the test drug once a day for 17 days in a row. The administration of substances that induce the Parkinson's disease model began on the 6th day. In the control group, mice were injected subcutaneously with saline at a dose of 10 ml per 1 kg of body weight; the remaining animals received MPTP subcutaneously at a dose of 25 mg per 1 kg of body weight once a day for 5 days.

Behavioral tests were performed on days 11, 14 and 17. The mouse was carefully placed on the rod (diameter 8 mm, height 55 cm) with its head on its embossed upper end. We recorded the time required for the animal to roll over from a head-up position to a head-down position, which is the latency period (Tturnover), and the time interval from the moment it reaches the head-down position until the moment it reaches the lower end of the rod on all four paws, which is the time of descent. (T _{climb off} ). Durations greater than 30 s were recorded as 30 s. Each individual underwent this test 5 times, and the data obtained were averaged.

MPTP causes selective damage to dopaminergic neurons in the substantia nigra of the brain. The MPTP-induced neurodegenerative state in mice is the closest model of human Parkinson's disease in terms of pathological features and clinical manifestations. The main symptoms of Parkinson's disease are tremors at rest, muscle rigidity, hypokinesia, etc. The test with a rollover and dismounting from the rod allows you to evaluate the general activity and coordination of the animal.

3. Animal model to determine the pharmacodynamics of the anti-inflammatory effect of the studied drugs (1) Rheumatoid arthritis model: collagen-induced arthritis in mice

For this experiment, male DBA/1 mice with a body weight of 19-22 g were used. The animals were randomly divided into groups of 8 animals each: control group, model group, and group receiving a therapeutic agent. All mice, except control animals, underwent immunological sensitization, namely, on day 0, bovine collagen type II with complete Freund's adjuvant (CII-CFA) was administered by subcutaneous injection into the base of the tail at a dose of 10 mg per 1 kg of body weight, and on day 23 were administered by intraperitoneal injection of lipopolysaccharide (LPS) at a dose of 1.5 mg per 1 kg of body weight. The administration of the test drugs began on the 28th day. Mice in the control group and model group were given saline solution orally, while in the other group they were given the test drug once a day for 14 days in a row. After injection of lipopolysaccharide, the animals were examined daily to monitor the development and course of the disease. Starting from the moment the disease manifested itself in mice, that is, clinical symptoms of arthritis arose, its progression was assessed on a severity scale in points from 0 to 4 based on the severity of symptoms (redness, joint deformation). A score of 0 indicated no erythema or joint swelling; 1 erythema and mild swelling in the area of the tarsals or tarsal joints or in the area of the metatarsals and redness and swelling on one finger; 2 - mild erythema and swelling of the tarsal joints and metatarsal bones or redness and swelling of more than two fingers; 3 - moderate erythema and swelling of the tarsal joints, wrist joints and metatarsal bones; 4 - severe redness and swelling of all tarsal joints, wrist joints, metatarsal bones and fingers. The highest number of points is 4 for each limb, 16 for one individual.

(2) MOG-induced multiple sclerosis mouse model.

In this experiment, female mice of the C57BL/6 line with a body weight of 17-20 g were used. Five individuals were randomly selected for the control group. The remaining animals were immunosensitized on day 0 by subcutaneous injection into the dorsum of myelin-oligodendrocyte glycoprotein emulsion with complete Freund's adjuvant (MOG-CFA) at a dose of 10 mg MOG and 20 mg complete Freund's adjuvant per 1 kg of body weight, as well as on days 0 and 2 - intraperitoneal injection of pertussis toxin at a dose of 10 mcg per 1 kg of body weight. Mice in the control group and the model group of animals were given saline solution orally, and the remaining mice were given the test drug once a day for 24 days in a row. Approximately 12 days after immunization in immunized

- 10,045,761 mice developed symptoms of the disease. From the onset of the first signs of disease, the animals were carefully examined daily, their body weight was noted, and their condition was scored on a clinical symptom scale ranging from 0 to 4 to monitor the progression of the disease. A score of 0 indicated normal appearance with no obvious signs of disease; 1 - decreased tone of the tail and weakness of one hind limb; 2 - decreased tone of the tail, weakness of both hind limbs and a staggering gait; 3 - weakness and paralysis of one hind limb; 4 - weakness and paralysis of both hind limbs.

(3) MRL/lpr mouse model of systemic lupus erythematosus

Transgenic MRL/lpr mice homozygous for the Faslpr gene mutation may spontaneously develop lymphoid tissue hyperplasia. At about 10-14 weeks of age, these mice develop symptoms of systemic lupus erythematosus. Female MRL/lpr transgenic mice, 9 weeks old, were randomly divided into groups of 8 animals each: a control group and a group receiving a therapeutic agent. Mice in the control group were given saline solution orally, and the rest were given the test drug once a day for 4 weeks in a row. Once a week, all animals were assessed for the condition of the lymph nodes using a clinical signs scale ranging from 0 to 6. A score of 0 meant normal; 1 - the diameter of the node in one location on both sides is less than 1 cm; 2 - the diameter of the node in two locations on both sides is less than 1 cm; 3 - the diameter of the node in three locations on both sides is less than 1 cm; 4 - the diameter of the node in one localization on both sides is more than 1 cm and in two other localizations on both sides it is less than 1 cm; 5 - the diameter of the node in two localizations on both sides is more than 1 cm and in one other localization on both sides is less than 1 cm; 6 - the diameter of the node in three locations on both sides is more than 1 cm.

(4) Inflammatory bowel disease (IBD) model - dextran sulfate sodium (DSS)-induced colitis in mice

For this experiment, female C57 mice were used, 7-8 weeks old and weighing 18-20 g, which were randomly divided into groups of 8 animals each: control group, model group and group receiving a therapeutic agent. In the model group and the group receiving the therapeutic agent, on days 1 to 7, mice were given the high molecular weight polymer dextran sulfate sodium at a concentration of 2.5% in their drinking water. In the control group and in the model group, mice were administered orally saline, and the remaining animals were administered the test drug once a day for 30 days in a row. On day 31, mice were sacrificed by cervical dislocation. The abdominal cavity of each individual was opened and the mesentery was separated. A section of intestine was removed from the beginning of the ileocecal region to the anus. Samples were taken sequentially one after another in each group. The length of the colon was measured.

4. Animal model to determine the pharmacodynamics of the drugs being studied to counteract diabetes mellitus

Male NIH mice were used for this experiment. The animals were randomly divided into groups of 10 animals each: control group; model group, the group receiving the therapeutic agent. On the day the experiment began, all mice, except the control ones, were administered streptozotocin by intraperitoneal injection at a dose of 150 mg per 1 kg of body weight. The test drug was given for 10 consecutive days. On the 11th day, the eyeballs of the animals were removed and blood was taken to determine the glucose concentration.

5. Animal model to determine the pharmacodynamics of the analgesic effect of the drugs under study (1) Acetic acid-induced pain model in mice

Kunming mice (equally divided between males and females) with a body weight of 18-22 g were randomly divided into groups of 10 animals each: control group, model group and group receiving a therapeutic agent. Starting from the day when the mice were divided into groups, animals in the control group were administered intragastric distilled water in an amount of 20 ml per 1 kg of body weight once a day for 7 days in a row; the remaining animals were administered intragastrically with the corresponding agent also once a day for 7 days in a row. 1 hour after the last injection, all animals were given an intraperitoneal injection of 0.2 ml of a 0.6% acetic acid solution. The latent period of writhing was recorded (the time period from the moment the acid was administered until the onset of a behavioral response (characteristic writhing)), as well as the number of writhings within 20 minutes after the administration of acetic acid.

Injecting mice with chemicals such as acetic acid into the abdominal cavity irritates the peritoneum, causing persistent intermittent pain, which is manifested by characteristic movements: the animal either presses its abdomen to the bottom of the cage, then retracts it, arches its back and tightens its buttocks, stretches its hind limbs (vinegar writhing). ). The latent period of writhing (the time interval between the administration of acetic acid and the occurrence of writhing) and their number over a certain period of time characterize the degree of pain. The shorter the specified latent period of writhing and the more writhing, the stronger the pain.

(2) Nitroglycerin-induced migraine model in rats

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Male Sprague Dawley (SD) rats weighing 180-220 g were randomly divided into groups of 8 animals each: control group, model group and group of individuals receiving a therapeutic agent. Administration began on the day of division into groups. Animals from the control group and from the model group were administered intragastric distilled water once a day for 28 consecutive days. The remaining rats were administered intragastrically with the test drug once a day for 28 consecutive days. 30 minutes after the last injection, all animals, except control animals, were administered nitroglycerin by subcutaneous injection into the right shoulder at a dose of 10 mg per 1 kg of body weight. The time interval from the moment of administration of nitroglycerin to the appearance of redness of the ears and the duration of this redness, as well as how many times within 40-45 minutes after the administration of nitroglycerin the animal rubs its head were recorded. In addition, the content of serotonin (5-hydroxytryptamine, abbreviated as 5-HT) in brain tissue was determined by fluorescence spectrophotometry (fluorescence excitation wavelength 356 nm; emission wavelength 483 nm). The results of these measurements were expressed in nanograms per 1 g of brain weight.

Migraine is a manifestation of dysfunction of blood vessels and nerves, caused by the interaction of the mechanisms of these two structures. Nitroglycerin causes hypersensitivity of the nerve fibers of the trigeminal nerve and dilation of the vessels of the meninges, initiating neurogenic inflammation and activating the functioning of neurons of the hypothalamus, brain stem and spinal cord; all this manifests itself as a migraine. Nitroglycerin-induced migraine in animals, proposed in 1995, is now the classic model of this disorder. According to the pathogenesis of nitroglycerin-induced migraine, the time of ear redness associated with vasodilation is used to assess its severity; the number of head rubs associated with the sensation of pain, and the content of serotonin, which is a factor in sensitivity to pain in brain tissue. The longer the redness of the ears lasts, the more the animal rubs its head and the higher the 5-HT level, the higher the severity of the migraine.

(3) Migraine model in rats induced by electrical stimulation of the trigeminal ganglion

Male Sprague Dawley (SD) rats, 5 months old, weighing 200–240 g, were randomly assigned to groups of 10 each: control group, sham-operated group, model group, and therapeutic agent group.

Rats of the first three groups received distilled water orally, individuals of the fourth group received the tested drug. After administration, which lasted for 10 consecutive days, all animals, except control animals, were anesthetized by intraperitoneal injection of chloral hydrate at a dose of 350 mg per 1 kg of body weight. Each individual was fixed in a stereotaxic apparatus and a midline incision was made from above the head, cutting through the skin and muscle layer by layer until the skull was exposed in the middle of the sagittal suture. Using a dental drill, a hole was drilled at a distance of 3 mm posteriorly and 3 mm lateral to the bregma and the electrode was inserted into the trigeminal ganglion to a depth of 9.5 mm from the dura mater. Anesthesia continued after this operation. All surgical procedures were performed under sterile conditions. After stimulation, the electrodes were removed. Stimulation parameters: cycle 200 ms; amplitude 10 V; pulse width 5 ms during 10 min stimulation. In the group of rats with sham surgery, the animals only received electrodes, but did not undergo electrical stimulation. 7 minutes before stimulation, the animal was injected with Evans blue dye into the right femoral vein, and no later than 20 minutes after stimulation, perfusion and fixation were performed.

5 minutes after the end of stimulation, the left ventricle was perfused for 2 minutes. A craniotomy was performed, the whole brain was removed and fixed for immunohistochemical determination of the c-fos protein in the pathological area. The localization of the electrode was also determined and a section of the dura mater was isolated from the site of insertion of the electrode and from the corresponding place in the other hemisphere of the brain, washed with deionized water, laid out in a flat layer on a glass slide, dried at a temperature of 37°C for 15 minutes and fixed with 70% glycerol. . The fluorescence intensity of the indicated tissue areas on the stimulated side and on the other (control) side was measured using a confocal fluorescence microscope (fluorescence excitation wavelength 647 nm; emission wavelength 680 nm). The stimulated/control fluorescence intensity ratio, reflecting plasma protein extravasation (PPE), was calculated. Continuous coronal sections of frozen whole brain were taken (section thickness 10 μm); cells expressing c-fos were labeled for immunofluorescence analysis. Using a confocal microscope, 5 fields were randomly selected to determine the number of cells with c-fos expression on the stimulated side and on the other (control) caudal part of the trigeminal spinal tract nucleus, and the average value for these five fields was taken as the average number of cells with c-fos expression fos.

In patients with migraine, activation of the neurovascular system of the trigeminal nerve is of key importance in the occurrence of pain; Neuroinflammation of the meninges plays an important role in the formation and maintenance of migraine pain. When the trigeminal nerve branches in the dura mater are stimulated, vasoactive substances are released, causing meningeal vasodilation, extravasation of plasma components, mast cell degranulation, and platelet activation, resulting in migraine. In addition, released after pain

- 12 045761 howl stimulation neurotransmitters bind to their receptors on the cell membrane. Under the influence of the second messenger, the expression of the c-fos gene is initiated, that is, the corresponding messenger RNA is synthesized and translated to form the c-fos protein, which leads to long-term physiological effects in the body. Thus, during migraine, in the nucleus of the spinal tract of the trigeminal nerve and the raphe magnus nucleus, the number of cells in which messenger RNA and c-fos protein are formed increases.

Therefore, the severity of migraine is reflected by the amount of serum protein released from the dura mater and the number of cells expressing c-fos in the caudal part of the trigeminal spinal tract nucleus. The fewer these cells, the weaker the migraine.

6. Animal model to determine the pharmacodynamics of counteraction of the studied drugs to vascular dementia (1) Model of vascular dementia in mice with bilateral common carotid artery occlusion (BCCAo)

By bilateral occlusion of the common carotid arteries, global cerebral ischemia/perfusion is induced in mice, which models vascular dementia.

1.1. Dividing animals into groups and administering agents

In this experiment, male C57BL/6 mice with a body weight of 22 ± 2 g were used, which were randomly divided into groups of 10 animals each: sham operation group; a group in which animals underwent bilateral occlusion of the common carotid arteries for 30 minutes (abbreviated as 30-minute BCCAo); and a group receiving a therapeutic agent. After dividing into groups, mice in the sham operation group and the 30-min BCCAo group were administered intragastric distilled water once a day for 5 consecutive days, after which the mice in the 30-min BCCAo group underwent surgery for bilateral occlusion of the common carotid arteries. Mice in the group receiving the therapeutic agent were administered intragastrically with the test drug once a day for 5 consecutive days, after which they underwent surgery for bilateral occlusion of the common carotid arteries. Surgical intervention for VSCAo consisted of the following. The animals were anesthetized with sodium phenobarbital, incisions were made on both sides of the neck, the common carotid arteries were isolated and clamped for 30 minutes, then the clip that was compressing the vessel was removed and the wound was sutured. In the pseudo-surgery group, after isolating the common carotid arteries, they were not clamped and the incision was immediately sutured. 24 hours after the BCCAo operation, mice of all groups continued to be given the appropriate drugs or water according to the same regimen as before the operation for 23 consecutive days. On the 7th day after surgery, a dark avoidance test was performed, and on the 13th day, a Morris water maze test was performed to evaluate the effect of administration of a composition with mannuronic diacid on learning and memory. After behavioral tests, animals were sacrificed, and brain tissue was removed and fixed. The degree of damage to neurons in the hippocampus after bilateral occlusion of the common carotid arteries and the protective effect of a composition with mannuronic diacid in neurons were determined by methods such as hematoxylin and eosin staining.

1.2. Dark avoidance test

The dark avoidance test is used to evaluate learning and spatial memory in mice. Impaired orientation in space and spatial memory can only appear with damage to the hippocampus or adjacent areas of the brain. The dark avoidance test device exploits the instinctive tendency of mice to prefer dark places to lighted ones. The box is divided into two halves, one of which is illuminated and the other is not, by a partition with a small hole in the center. In the dark half, the bottom of the box is covered with copper mesh. When the mouse is on the dark half, an electric current is applied to the grid, and the animal, having received an electric shock, runs away to the light half of the box. The mice are trained for 24 hours, after which the test is repeated. The period of time from the moment the mouse is placed in the lighted half of the box until it moves to the dark half is called the latent period. The longer the latency period and the less often the animal makes mistakes, the better its ability to remember.

1.3. Morris water maze test

In the Morris water maze, the animal must learn to find an underwater platform by swimming. This test is mainly used to evaluate learning ability regarding spatial position and direction memory. The setup for this test consists of a cylindrical pool with a diameter of 80 cm and a height of 70 cm, in which there is a movable platform with a diameter of 8 cm. At the top is a digital video camera connected to a computer. Before the test, the pool is filled with clean water to a depth of 15 cm, the platform is located 0.5 cm below the surface of the water. Milk is added to the water so that it is cloudy and the platform is not visible. During the experiment, the position of the platform does not change. During the test, the following indicators were determined.

The navigation test is used to determine the mouse's ability to explore and remember a given space. This test was carried out for 4 days, starting from the 13th day after surgery for bilateral occlusion of the common carotid arteries. At first, the animal was given training: they were allowed into the pool once in the morning and in the evening, 8 times in total. The training consisted of placing the animal

- 13 045761 into the pool on half the arc of the western quadrant of the circle and immersed in the water with the head to the wall of the cylinder. If the mouse did not find the platform within 120 s, the experimenter directed it there and left it on the platform for 30 s so that the animal remembered the location. Using a camera, the path and time of searching and climbing onto the platform for each individual are observed and recorded and the latent period and swimming speed are determined. The escape latency period in the Morris water maze is the period of time from the moment the mouse is placed in the water until it finds the platform. The shorter this latency period, the better the animal's ability to remember.

The spatial search test is used to evaluate a mouse's ability to retain the location of a platform in memory after it has learned to find it. At the end of the navigation test after 1 day, the platform was removed, the mouse was placed in water at the same starting point, and the number of times it crossed the place where the platform was previously located was recorded. Data collection and processing were carried out using an automatic image recording and processing system.

(2) Model of vascular dementia in rats after middle cerebral artery occlusion (MCAO)

By occluding the middle cerebral artery, focal cerebral ischemia is caused in rats, which models vascular dementia.

2.1. Division into groups and introduction of agents

In this experiment, male Wistar rats were used, which were divided into 4 groups of 10 animals each: control group; pseudo-operation group, model group (MCAO group); group receiving the therapeutic agent. In the control group, sham operation group, and MCAO group, rats were given distilled water orally; rats in the therapeutic agent group were given oligosaccharides from alginic acid. After 7 days of administration of these agents, all animals, except those from the first control group, were anesthetized by intraperitoneal injection of chloral hydrate at a dose of 350 mg per 1 kg of body weight and fixed on a surgical table in a position on the left side. Under an operating microscope, the skin was incised along the middle of the line connecting the edge of the external auditory canal and the angle of the palpebral fissure to expose the zygomatic arch. The connection with the mandible was expanded with a small distractor. A 2x2 mm hole was made at the base of the skull. The dura mater was incised to expose the middle cerebral artery, and on one side the vessel was coagulated with high-frequency electrocautery, which caused local ischemia (in the sham operation group, the middle cerebral artery was exposed but not coagulated). The incision was sutured in layers. During the operation, the temperature in the room was strictly controlled, maintaining 24-25°C. After surgery, the animals continued to be given the therapeutic agent or distilled water in the same regimen as before surgery. On the 11th day after surgery, each group of individuals was tested in the Morris water maze.

In the Morris water maze, all rats were trained once a day for 5 consecutive days. For each individual, the length of time it took to find the platform was recorded (the escape latency in the navigation test). Those rats that could not find the platform within approximately 120 s were directed to the platform in a straight line and allowed to stand on it for 30 s to allow the animal to learn and remember its location. At the end of the navigation test, 1 day later, after removing the platform, the rat was placed in water at the same starting point and it was recorded how long it would take for the first time to be at the place where the platform was, and how many times it would cross this place (spatial search test ). Each animal was assessed for learning and memory abilities. The latent period of escape in the Morris water maze, that is, the period of time from the moment the animal is placed in the water until the moment when it finds the platform, the shorter the latent period, the better the memory of a given individual.

Hereinafter, the advantages of the present invention are illustrated by non-limiting examples. The specific materials and quantities thereof, as well as other experimental conditions used in the examples, should not be construed as limiting the invention. In this document, all meanings of parts, shares, percentages, etc. calculated by weight unless otherwise stated.

Example 1.

Stage 1. Preparation of a mixture of oligosaccharides from alginic acid

A solution of sodium alginate (5 kg) in water with a concentration of about 10% was prepared and the pH was adjusted to about 3.0 by adding dilute hydrochloric acid. The resulting solution was heated to 80°C and stirred. The reaction was left at this temperature for 10 hours. After this time, the solution was cooled to room temperature and the pH was adjusted to 9.0 by adding NaOH, after which dilute hydrochloric acid was added to pH 3.2. After this, the solution was centrifuged at 5000 rpm for 10 min. The supernatant was collected, concentrated by rotary evaporation and dried in vacuo to yield 1500 g of intermediate.

In fig. Figure 1 shows the NMR spectrum of this intermediate. NMR analysis was carried out as follows. Sample preparation: 30 mg of the material to be analyzed was weighed out and dissolved in 0.5 ml of heavy water (D ₂ O), then lyophilized; dissolved the lyophilisate by adding 0.5

- 14 045761 ml of heavy water and lyophilized again; finally, the powder obtained as a result of lyophilization was dissolved in the required amount of heavy water, placed in a test tube for NMR spectroscopy, and an analysis solution with a concentration of 100 mg/ml was obtained; A 0.01% (w/v) solution of trimethylsilyl tetradeuteropropionic acid (TSP) sodium salt was used as an internal standard. Data collection and processing: using a 400M Fourier spectrometer, one-dimensional proton spectra were recorded at a temperature of 60°C. Pulse magnitude 45°, interval 4 s, relaxation time 1 s, accumulation 20-fold, chemical shift from -2 ppm. up to 10 ppm After data collection, a Fourier transform was performed to obtain a one-dimensional proton spectrum; the signal for TSP was taken as 0.00 ppm.

In fig. 1 shows that the intermediate product contained a region with mannuronic acid (fragment M, chemical shift 5.1 ppm) and a region with guluronic acid (fragment G, chemical shift 5.5 ppm), as well as a chimeric region with mannuronic and guluronic acids (MG fragment, chemical shift 5.3 ppm). 500 g of the intermediate product were weighed and dissolved in distilled water, so that 5 liters of solution was obtained. The pH of this solution was adjusted to 6.5 using NaOH and heated in a water bath, maintaining the temperature at 75°C. Ozone was passed through it, selecting the gas flow rate at the outlet of the oxygen cylinder and the power of the ozone generator so that ozone entered the specified solution at a rate of 8 g/h. After 4 hours of the reaction, the supply of ozone was stopped and such an amount of water was added so that the concentration of the solution became about 10%. The resulting solution was passed through an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the retentate was collected. The collected liquid was concentrated using a rotary evaporator and dried in vacuum; the result was 350 g of product A.

Stage 2. Analysis of the ratio and structures of oligosaccharides with different degrees of polymerization in product A containing oligomeric sugar diacids from alginic acid

Exactly 100 mg of the dried product A obtained as described above, containing oligomeric sugar diacids from alginic acid, was weighed out and dissolved in water at a concentration of 10 mg/ml. This solution was passed through a filtration membrane (0.22 μm), thus obtaining a solution for analysis. The relative amounts of oligosaccharides with different degrees of polymerization in the mixture were determined using size exclusion chromatography on Superdex columns (GE Healthcare Co.) coupled with multi-angle laser light scattering (MALS, Wyatt Co.). The conditions were as follows.

Chromatography column - Superdex peptide 10/300G1

Mobile phase - NaCl solution 0.1 mol/l

Injected sample volume - 10 µl

Flow rate - 0.3 ml/min

The following results were obtained. Components from disaccharides to decasaccharides (dp2-dp10) were present in relative amounts: dp2 - 18%, dp3 -24%, dp4 - 23%, dp5 - 14%, dp6 - 8%, dp7 7%, dp8 - 2%, dp9 - 2% and dp10 - 2%.

Step 3. Analysis of the structure of oligosaccharides with different degrees of polymerization in product A, containing oligomeric sugar diacids from alginic acid, by liquid chromatography with mass spectrometry (LC-MS)

Experimental conditions:

Column Superdex peptide 10/300G1

Mobile phase - 20% methyl alcohol + 80% ammonium acetate (NH ₄ Ac) (80 mmol/l)

Flow rate - 0.1 ml/min

The temperature in the column is 2°C ± 0.8°C.

Mass spectrometry conditions:

Agilent 6540 QTO tandem quadrupole time-of-flight mass spectrometer;

Ion source - ESI electrospray (voltage 120 V)

Mode - negative ions

Mass range m/z - 100-1000.

In fig. 1-3 show the mass spectra of oligosaccharides with different degrees of polymerization. The signal peak pattern of these mass spectra confirms the molecular structure of the oligosaccharides in product A as the structure depicted by the general formula (III). Below is the table. 1, which shows the structures and their corresponding signals.

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Table 1. Structure of six diacids of oligosaccharides with different degrees of polymerization in product A and the corresponding mass/charge ratios

No. Structural formula Chemical formula Mass/charge ratio (m/z) n=1 [M-1]- n=2 [M-1]’ n=3 [M-1]’ n=4 [M-1]’ n=5 [M-1]’ n=6 [M-1]’ n=7 [M-2] ² ' n=8 [M-2] ² ' n=9 [M-2] ² ' 1 g noos - hJ °* ⁰ 1 Ηο-7^^“θΗ [°no ¹ ^on (SbN ₈ Ob)pSbNyuO ₈ p=1-9 385 561 737 913 1089 1265 720 808 896 2 g NOOS, (C ₆ H ₈ O ₆ ) _P S ₅ H ₈ O ₇ p = 1-9 355 531 707 883 1059 1235 705 793 881 3 (SbN ₈ Ob) pS ₅ N ₈ O ₇ p = 1-9 355 531 707 883 1059 1235 705 793 881 4 g NOOS - (SbN ₈ Ob) pS ₄ N _b Ob n = 1-9 325 501 677 853 1029 1205 690 778 866 5 (SbN ₈ Ob) pS ₄ N _b Ob n = 1-9 325 501 677 853 1029 1205 690 778 866 6 (SbN ₈ About) _p SzN ₄ O5 p = 1-9 295 471 647 823 999 1175 675 763 851

From the results of the mass spectrometric analysis described above, it follows that the mannuronic or guluronic acid residue at the reducing end of the polysaccharide chain in product A is oxidized to a sugar acid structure (see general formula IV); this structure may be that of mannaric or guluronic acid containing six carbon atoms (m+m'=3), which is contained in an amount of about 10%-30%, or the decarboxylation product of mannaric or guluronic acid, that is, a sugar diacid containing five atoms carbon (m+m'=2), which is present in quantity (30-50%) and sugar diacid with four carbon atoms (m+m-l), the content of which is 30%-40%.

Step 4. NMR analysis of guluronic acid content in product A containing oligomeric sugar diacids from alginic acid

Sample preparation: 50 mg of the material to be analyzed was weighed out and dissolved in 0.5 ml of heavy water (D ₂ O), then lyophilized; to dissolve the lyophilisate, 0.5 ml of heavy water was added to it and lyophilized again; finally, the powder obtained as a result of lyophilization was dissolved in the required amount of heavy water, placed in a test tube for NMR spectroscopy, and an analysis solution with a concentration of 100 mg/ml was obtained; A 0.01% (w/v) solution of trimethylsilyl tetradeuteropropionic acid (TSP) sodium salt was used as an internal standard.

Data collection and processing: using a 400M Fourier spectrometer, one-dimensional proton spectra were recorded at room temperature (pulse magnitude 45°, interval 4 s, relaxation time 1 s, accumulation 20-fold, chemical shift from -2 ppm to 10 m .d.). After data collection, a Fourier transform was performed to obtain a one-dimensional proton spectrum; the signal for TSP was taken as 0.00 ppm. In fig. 5 shows the 1H NMR spectrum of product A. FIG. 5 multiplet with a chemical shift of 4.6 ppm. signal of the proton at the C-1 position of mannuronic acid (M); multiplet with a chemical shift of 5.0 ppm. proton signal at position C-1 of guluronic acid (G); multiplet with a chemical shift of 4.9 ppm. - proton signal at position C-1 of the region including mannuronic and guluronic acid (MG). The content of guluronic acid was calculated using the formula

I5 o + O.BS g

G% = x 100% ^5.0 + U.6 + U.9

In this formula, 1 ₄ , ₆ , I ₅ , ₀ and I ₄ , ₉ are the values of the integral intensity of the proton signal at the C-1 position of mannuronic acid (M), guluronic acid (G) and the chimeric region including mannuronic and guluronic acid (MG ) respectively. According to calculations, the content of guluronic acid in product A is 30%.

Example 2.

Weighed 100 g of commercially available sodium alginate (purchased from Sinopharm Reagent Co., CAS No. 9005-38-3, chemically pure, quality control of chemical reagents in Shanghai), added distilled water, stirred until homogeneous, and after swelling, adjusted the volume of the solution to 1 l. The pH of the resulting solution was adjusted to 4.0 using NaOH. The reaction was carried out at room temperature (25 °C). Ozone was passed through the solution, selecting the gas flow rate at the outlet of the oxygen cylinder and the power of the ozone generator so that ozone entered the specified solution at a rate of 1 g/h. After 10 hours of the reaction, the supply of ozone was stopped and such an amount of water was added so that the concentration of the solution became about 15%. The resulting solution was passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the retentate was collected. The collected liquid was concentrated using a rotary evaporator and dried in vacuum; the result was 80 g of product B.

Relative amount of oligosaccharides with different degrees of polymerization in product B

- 16 045761 was determined by size exclusion chromatography on Superdex columns (GE Healthcare Co.) combined with multi-angle laser light scattering (MALS, Wyatt Co.). The measurements were carried out in the same way as described in the corresponding section of Example 1. The following results were obtained. Components from disaccharides to decasaccharides (dp2-dp10) were present in relative amounts: dp2 - 25%, dp3 24%, dp4 - 18%, dp5 - 13%, dp6 - 10%, dp7 -5%, dp8 - 2%, dp9 - 2% and dp10 - 1%.

The guluronic acid content of product B was 50% as determined by NMR analysis using a 400M FTIR spectrometer at 60°C. The measurements were carried out in the same way as described in the corresponding section of Example 1. In FIG. 6 shows proton NMR spectra; it can be seen that the integrated signal intensities (area under the peak) for mannuronic acid (M, chemical shift 4.6 ppm) and for guluronic acid (G, chemical shift 5.0 ppm) are relatively close, while as the integrated signal intensity for the chimeric region, including both mannuronic and guluronic acids (MG, chemical shift 4.9 ppm), is low. According to the above formula for calculating the content of guluronic acid (G), it is 50%.

Example 3.

We weighed 100 g of the intermediate product according to example 1. Added water, adjusted the pH of the resulting suspension to alkaline, adding NaOH so that the powder was completely dissolved. Water was added to a volume of 1 liter and the pH was adjusted to 2.95 by adding HCl. A white precipitate appeared, which was removed by centrifugation. The supernatant was collected. It was diluted with distilled water to a volume of 1.5 liters. The resulting solution was adjusted to pH 9.0 by adding NaOH. The reaction was carried out in a water bath at a temperature of 45°C. Ozone was passed through the solution, selecting the gas flow rate at the outlet of the oxygen cylinder and the power of the ozone generator so that ozone entered the specified solution at a rate of 3 g/h. After 2 hours of the reaction, the supply of ozone was stopped and such an amount of water was added so that the concentration of the solution became about 5%. The resulting solution was passed through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the retentate was collected. The collected liquid was concentrated using a rotary evaporator and dried in vacuum; the result was 60 g of product C.

The relative amounts of oligosaccharides with different degrees of polymerization in product C were determined by size exclusion chromatography on Superdex columns (GE Healthcare Co.) coupled with multi-angle laser light scattering (MALS, Wyatt Co.). The measurements were carried out in the same way as described in the corresponding section of Example 1. The following results were obtained. Components from disaccharides to decasaccharides (dp2-dp10) were present in relative amounts: dp2 - 9%, dp3 - 21%, dp4 - 27%, dp5 - 18%, dp6 - 13%, dp7 - 6%, dp8 - 3%, dp9 - 2% and dp10 - 1%.

The content of guluronic acid in product C was 10% according to NMR analysis using a 400M Fourier transform spectrometer at a temperature of 60°C. The measurements were carried out in the same way as described in the corresponding section of example 1. The results obtained are presented in Fig. 7; it can be seen that the integral signal intensity for mannuronic acid (M, chemical shift 4.6 ppm) is 13 times higher than the integral signal intensity for guluronic acid (G, chemical shift 5.0 ppm), and the integral signal intensity for chimeric region including both mannuronic and guluronic acids (MG, chemical shift 4.9 ppm) is close to the value for guluronic acid. According to the above formula for calculating the content of guluronic acid (G), it is 10%.

Example 4. Evaluation of the pharmacological activity of a composition of oligomeric sugar diacids from alginic acid in comparison with the hexamer of mannuronic acid

Preparation of samples

1. Preparation of mannuronic acid hexamer

20 g of mannuronic acid hexamer were prepared by the methods described in examples 1 and 2 of the previous patent 200580009396.5.

In table 2 below shows the content of various oligosaccharides and guluronic acid in products A, B and C obtained in Examples 1, 2 and 3 of this document described above.

2. Obtaining product D

A product with a high content of guluronic acid was obtained by the method described in example 2 above. The starting material was sodium alginate with a high content of guluronic acid, provided by Qingdao Haizhilin Biotechnology Development Co., Ltd. The production method was the same as described in the relevant part of Example 2. Namely, 500 g of sodium alginate powder with a high content of guluronic acid was mixed with distilled water until homogeneous; after swelling, water was added to a volume of 5 l and the pH was adjusted by adding NaOH. The reaction was carried out at room temperature (25°C). Ozone was passed through the solution, selecting the gas flow rate at the outlet of the oxygen cylinder and the power of the ozone generator so that ozone entered the specified solution at a rate of 1 g/h. After 12 hours of the reaction, the supply of ozone was stopped and such an amount of water was added so that the concentration of the solution became about 15%. The resulting solution was passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the retentate was collected. The collected liquid was concentrated using a rotary evaporator and dried in vacuum; the result was 350 g of product D.

The relative amounts of oligosaccharides with different degrees of polymerization in product D were determined by size exclusion chromatography on Superdex columns (GE Healthcare Co.) coupled with multi-angle laser light scattering (MALS, Wyatt Co.). The measurements were carried out in the same way as described in the corresponding section of Example 1. The following results were obtained. Components from disaccharides to decasaccharides (dp2-dp10) were present in relative amounts: dp2 - 18%, dp3 - 26%, dp4 - 20%, dp5 - 15%, dp6 - 8%, dp7 - 7%, dp8 - 3%, dp9 - 2% and dp10 - 1%.

The guluronic acid content of product B was 60% as determined by NMR analysis using a 400M FTIR spectrometer at 60°C. The measurements were carried out in the same way as described in the corresponding section of Example 1. The proton NMR spectra are presented in Fig. 8; it can be seen that the integral signal intensity for guluronic acid (G, chemical shift 5.0 ppm) exceeds the integral signal intensity for mannuronic acid (M, chemical shift 4.6 ppm), and the integral signal intensity for the chimeric region , including both mannuronic and guluronic acids (MG, chemical shift 4.9 ppm) is less. According to the above formula for calculating the content of guluronic acid (G), it is 60%.

Table 2. Contents of various oligosaccharides and guluronic acid in compositions of oligomeric diacids from alginic acid

Contents Product Disaccharides Trisaccharides Tetrasaccharides Pentasaccharides Hexasaccharides Heptasaccharides Octasaccharides Nonasaccharides Decasaccharides Guluronic acid A 18% 24% 23% 14% 8% 7% 2% 2% 2% thirty% IN 25% 24% 18% 13% 10% 5% 2% 2% 1% 50% WITH 9% 21% 27% 18% 13% 6% 3% 2% 1% 10% D 18% 26% 20% 15% 8% 7% 3% 2% 1% 60%

To study pharmacodynamics, samples weighing 10 g of each of products A, B, C, D and the mannuronic diacid hexamer were taken. The pharmacological activity of four compositions of oligomeric sugar diacids from alginic acid according to this invention was compared with the activity of a hexamer of mannuronic diacid by the methods described in sections Animal model to determine the pharmacodynamics of counteraction of the studied drugs for Alzheimer's disease, Animal model to determine the pharmacodynamics of counteraction of the studied drugs for Parkinson's disease, Model for animals to determine the pharmacodynamics of the anti-inflammatory effect of the studied drugs, an animal model to determine the pharmacodynamics of the anti-diabetes mellitus of the studied drugs, an animal model to determine the pharmacodynamics of the analgesic effect of the studied drugs, an animal model to determine the pharmacodynamics of the resistance of the studied drugs to vascular dementia in this document.

1. Determination of the pharmacodynamics of the resistance of the studied drugs to Alzheimer's disease

In these experiments, the platform detection latency in the model group was significantly longer than in the sham group, indicating that the disease model used was successful. Compared to the model group, the latency period for platform detection was significantly shorter in all individuals treated with the therapeutic agent.

One day after completion of the navigation test, the platform was removed and a spatial search test was performed. The number of times the animal crossed the area where the platform was was recorded; We calculated the ratio (in percentage) of the distance that the animal swam in the quadrant where the platform was located to the total distance covered by it in the entire pool. Based on these data, the learning ability and memory of this individual were assessed. The results of these experiments showed that, compared with the pseudo-surgery group, the number of platform crossings was significantly lower in the model group, and the rate was significantly higher in the group receiving the therapeutic agent with this pseudo-surgery (see Fig. 9). The ratio (in percentage) of the distance that the animal swam in the quadrant where the platform was located to the total distance covered by it in the entire pool varied in the indicated groups in the same way as the number of crossings of the platform location. Compared with the group with pseudo-operation in the model group, the ratio (in percentage) of the distance that the animal swam in the quadrant where the platform was located to the total distance covered by it in the entire pool was significantly less, and in the group receiving the drug agent, this the figure was significantly higher (see Fig. 10).

The results of these experiments showed that the pharmacodynamic activity of products A, B and C is higher than the hexamer of mannuronic diacid, therefore, the composition of oligosaccharides with a certain content of guluronic acid, in which the ratio of the amount of disaccharides to the amount of hexasaccharides exceeds 60%, has a synergistic effect. And the composition of oligosaccharides D, in which the content of guluronic acid was higher, has lower activity.

2. Determination of the pharmacodynamics of the resistance of the studied drugs to Parkinson's disease.

In this experiment, the latent period and duration of dismounting from the rod in animals in

- 18 045761 in the model group were significantly longer than in individuals of the control group. And in the group receiving the therapeutic agent, these indicators were to one degree or another lower than in individuals from the model group, and the pharmacodynamic activity of products A, B and C was higher than that of the mannuronic diacid hexamer with a single degree of polymerization, which was previously thought the most active. Without being bound by any theory, we believe that the guluronic acid content of the composition and the ratio of disaccharides to hexasaccharides significantly influence the activity of the product, and if the guluronic acid content is too high, the activity of the composition is reduced (see Figs. 11 and 12 ).

3. Determination of the pharmacodynamics of the anti-inflammatory effect of the studied drugs (1) Model of collagen-induced arthritis in mice

In this experiment, the model group had distinct symptoms of arthritis compared to the control group: mild erythema and swelling of the tarsal joints, wrist joints, and metatarsal bone areas. The clinical condition score reached 6 points, which indicated the success of the model used. In animals treated with a therapeutic agent, the severity of arthritis symptoms was to one degree or another less than in animals from the model group. The introduction of products A, B and C significantly delayed the onset of the disease compared to the mannuronic diacid hexamer, an oligosaccharide with a single degree of polymerization. Clinical scores were also lower for products A, B and C compared to mannuronic diacid hexamer. Consequently, the pharmacodynamic activity of products A, B and C is higher than that of the mannuronic diacid hexamer. As for product D, in this case the disease began earlier and the clinical picture score was greater than in the case of mannuronic diacid hexamer, which means that the activity of product D was weaker. This suggests that the guluronic acid content of the composition and the ratio of disaccharides to hexasaccharides significantly influence the activity of the product, and if the guluronic acid content is too high, the activity of the composition is reduced.

(2) MOG-induced multiple sclerosis mouse model

In this test, the majority of mice in the model group showed weakness and paralysis of both hind limbs (in contrast to the healthy control group). The average clinical picture score in this group reached 3 points, indicating the success of the disease model used. In mice treated with a therapeutic agent, the progression of inflammation was, to varying degrees, weaker than in the model group. The clinical picture scores for animals receiving products A, B, and C were lower throughout the experiment and at the end point than for those animals receiving mannuronic diacid hexamer; this indicated that the pharmacodynamic activity of products A, B and C was higher than that of the mannuronic diacid hexamer. As for product D, the assessment of the clinical picture in individuals receiving it throughout the experiment and at the end point was slightly higher; this indicated that its activity was the weakest. This suggests that the guluronic acid content of the composition and the ratio of disaccharides to hexasaccharides significantly influence the activity of the product, and if the guluronic acid content is too high, the activity of the composition is reduced.

(3) MRL/lpr mouse model of systemic lupus erythematosus

In transgenic mice of the MRL/lpr line, starting from the 10th week of life, a disease spontaneously developed, manifested, in particular, by swelling of the lymph nodes, and the assessment of the condition of the lymph nodes in points increased over time, which indicated the success of the disease model used and rapid progression of this disease. In the group of mice treated with a therapeutic agent, the progression of the disease was weakened to varying degrees compared to the model group. Products A, B and C, compared with mannuronic diacid hexamer, significantly delayed the onset of disease in mice, and lymph node scores were also lower for these three products, indicating greater pharmacodynamic activity. For product D, the onset of disease was earlier and the lymph node score was higher than for mannuronic diacid hexamer, indicating a weaker activity of this product. Therefore, the guluronic acid content of the composition and the ratio of the amount of disaccharides to the amount of hexasaccharides significantly influence the activity of the product, and if the guluronic acid content is too high, the activity of the composition is reduced.

(4) Dextran sulfate sodium (DSS)-induced colitis model in mice

After completing this test, the model group of animals had significantly less colon length than healthy controls due to inflammation caused by dextran sulfate sodium, and most of these mice lost body weight. Of the animals in the model group, almost half later died, indicating very severe intestinal inflammation. In individuals receiving the therapeutic agent, compared with the model group, intestinal inflammation was to one degree or another weaker, which was manifested in less shortening of the colon and higher survival. In fig. 13a and 13b it can be seen that in the case of products A, B and C, the length of the colon

- 19 045761 and mouse survival were higher than in the case of mannuronic diacid hexamer, indicating higher pharmacodynamic activity of all products A, B and C compared to mannuronic diacid hexamer. As for product D, colon length and mouse survival were slightly less than with mannuronic diacid hexamer, indicating weaker activity of this product. The results of this experiment are consistent with those described above, indicating that the guluronic acid content of the composition and the ratio of disaccharides to hexasaccharides significantly influence the activity of the product, and if the guluronic acid content is too high, the activity of the composition is reduced.

4. Determination of the pharmacodynamics of the drugs being studied to counteract diabetes mellitus

This test compared a model group with a healthy control group, and the animal model group had significantly higher postprandial blood glucose levels, indicating that the disease model used was successful. In the group of animals receiving the therapeutic agent, blood glucose levels were significantly lower than in the model group. At the same time, the pharmacodynamic activity of each of products A, B and C was higher, and that of product D was lower than that of the mannuronic diacid hexamer. The results of this experiment are consistent with those described above, indicating that the guluronic acid content of the composition and the ratio of disaccharides to hexasaccharides significantly influence the activity of the product. However, if the guluronic acid content is too high, the activity of the composition is reduced (see Fig. 14).

5. Determination of the pharmacodynamic anti-pain activity of the studied drugs (1) Acetic acid-induced pain model in mice

In this experiment, the latent period for the occurrence of writhing in mice of the model group was significantly shorter and the number of writhings over a certain period of time was greater than in animals of the control group, which indicated the success of the model used. In mice that were injected with a therapeutic agent, the latent period for the occurrence of writhing was much longer and the number of writhings was significantly less than in the animals of the model group. In the case of each of products A, B and C, the latency period for the occurrence of cramps was longer and their quantity was less than in the case of using the mannuronic acid hexamer, which indicated a higher pharmacodynamic activity of each of these products compared to the mannuronic acid hexamer. In the case of product D, the latent period for the occurrence of cramps was shorter and their number was slightly higher than in the case of mannuronic acid hexamer, indicating a weaker activity of product D. The results of this experiment are consistent with those described above, indicating that the content of guluronic acid in the composition and the ratio of the amount of disaccharides to the amount of hexasaccharides significantly affect the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition is reduced (see Fig. 15 and 16.).

(2) Nitroglycerin-induced migraine model in rats

In rats, after a subcutaneous injection of nitroglycerin, redness of the ears appears after approximately 3 minutes, which lasts for about 2.5 hours. In animals of the model group, the number of head rubbings 30-45 minutes after the onset of this symptom was significantly greater than in healthy animals of the control group. In animals receiving the therapeutic agent, ear redness appeared much later and lasted less, and the number of head rubs in 30-45 minutes was less than in animals of the model group. Products A, B, and C had fewer head rubs than mannuronic diacid hexamer, indicating greater pharmacological activity for each of Products A, B, and C compared to mannuronic diacid hexamer. In the case of product D, the number of head rubs in rats was slightly greater than in the case of mannuronic acid hexamer, indicating a weaker activity of product D. The results of this experiment are consistent with those described above, indicating that the content of guluronic acid in the composition and the ratio of the amount disaccharides to the amount of hexasaccharides significantly affect the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition is reduced (see Fig. 17.).

(3) Trigeminal ganglion electrical stimulation-induced migraine model in rats

Electrical stimulation of the trigeminal ganglion causes extravasation of plasma protein in the dural vessels in rats. In animals of the model group, extravasation of blood plasma protein was significantly more pronounced and the number of cells with c-fos expression was much greater than in healthy animals in the control group and in animals from the sham-operation group. In animals receiving the therapeutic agent, extravasation of plasma protein was significantly less pronounced and the number of cells expressing c-fos was much lower than in animals of the model group. In the case of products A, B, and C, the number of cells expressing c-fos was lower than in the case of mannuronic diacid hexamer, indicating higher pharmacological activity of each of products A, B, and C compared to mannuronic diacid hexamer. In the case of product group D, the number of cells expressing c-fos was slightly higher than in the case of mannuronic acid hexamer, indicating weaker activity of product D. The results of this experiment are consistent with those described above, indicating that the content guluronic acid in the composition and the ratio of the amount of disaccharides to the amount of hexasaccharides significantly affect the activity of the product. However, if the guluronic acid content is too high, the activity of the composition is reduced (see Fig. 18).

6. Determination of the pharmacodynamics of counteraction of the studied drugs to vascular dementia (1) Model of vascular dementia caused by bilateral occlusion of the common carotid arteries in mice (BCCAo)

1.1. Dark avoidance test results

The experiment compared a model group of mice and a group of sham-operated mice. In the model group, the latent period of dark avoidance was significantly shorter and the number of errors was greater, indicating that in animals with bilateral occlusion of the common carotid arteries, the ability to remember was significantly reduced and, therefore, vascular dementia was modeled successfully. In the group of animals receiving the therapeutic agent, the latent period of dark avoidance was significantly longer and the number of errors was much less than in mice of the model group. In the case of products A, B and C, the latent period of dark avoidance was longer than in the case of mannuronic diacid hexamer, and the number of errors for products A, B and C was less than for mannuronic diacid hexamer, indicating higher pharmacological activity of the products A, B, and C compared to the mannuronic diacid hexamer. For product D, the dark avoidance latency was slightly shorter than for mannuronic acid hexamer, and the error rate was also slightly higher, indicating weaker activity for product D. The results of this experiment are consistent with those described above, indicating that the content guluronic acid in the composition and the ratio of the amount of disaccharides to the amount of hexasaccharides significantly affect the activity of the product. However, if the guluronic acid content was too high, the activity of the composition was reduced (see FIGS. 19 and 20).

1.2 Morris water maze test results

During the experiment, the latent period of escape in the Morris water maze was significantly longer in the model group than in the sham group, indicating that the model of BCCAo-induced vascular dementia functions successfully. In the group of animals receiving the therapeutic agent, the latent period of rescue was significantly shorter than in the mice of the model group. Products A, B, and C had shorter rescue latencies than mannuronic diacid hexamer, indicating higher pharmacological activity of products A, B, and C compared to mannuronic diacid hexamer. In the case of product D, the latent period of rescue was slightly longer than in the case of mannuronic acid hexamer, indicating weaker activity of product D. (See Fig. 21.)

4 days after the navigation test, the platform was removed and a spatial search test was performed, determining how many times the animal crossed the place where the platform was. Compared with the sham operation group, the number of crossings of the previous platform location by mice of the model group was significantly less, indicating that bilateral occlusion of the common carotid arteries leads to a significant decrease in the ability to remember, while in the group of animals receiving a therapeutic agent, the number There were more intersections with the former location of the platform. In the case of products A, B, and C, the number of intersections of the previous platform location was greater than in the case of the mannuronic diacid hexamer, indicating a higher pharmacological activity of products A, B, and C compared to the mannuronic diacid hexamer. In the case of product D, the number of crossings of the previous platform location was slightly less than in the case of mannuronic acid hexamer, indicating weaker activity of product D. (See Fig. 22.) (2) Effect of the tested drugs in rats with vascular dementia, caused by middle cerebral artery occlusion (MCAO)

During the experiment, the latent period of escape in the Morris water maze was significantly longer in the model group than in the sham group, indicating that the MCAO-induced vascular dementia model functioned successfully. In the group of animals receiving the therapeutic agent, the latent period of rescue was significantly shorter than in the mice of the model group. Products A, B, and C had shorter rescue latencies than mannuronic diacid hexamer, indicating higher pharmacological activity of products A, B, and C compared to mannuronic diacid hexamer. In the case of product D, the latent period of rescue was slightly longer than in the case of mannuronic acid hexamer, indicating weaker activity of product D.

1 day after the navigation test, the platform was removed and a spatial search test was performed, determining how many times the animal crossed the place where the platform was in 2 minutes. The number of crossings of the previous platform location in the model group was significantly less than in the sham operation group, indicating that occlusion of the middle cerebral artery leads to a significant decrease in the ability to remember that time, as in the group of animals receiving a therapeutic agent. , the number of crossings of the platform's previous location was higher. In the case of products A, B, and C, the number of crossings of the previous platform location was higher than in the case of the mannuronic diacid hexamer, indicating higher pharmacological activity of products A, B, and C compared to the mannuronic diacid hexamer. In the case of product D, the number of crossings of the previous platform location was slightly less than in the case of mannuronic acid hexamer, indicating weaker activity of product D.

Claims (15)

1. Pharmaceutical composition of oligomeric sugar diacids derived from alginic acid, where the composition contains a mixture of mannuronic acid and guluronic acid of formula (IV), or their pharmaceutically acceptable salts: Formula (IV) where n is an integer selected from 1 to 9, m is selected from 0, 1 or 2, m' is selected from 0 or 1 and the total mass of oligomeric sugar diacids derived from alginic acid, in which n= 1-5, constitutes at least 60% of the total weight of the composition; wherein the total mass of guluronic acid is 0.1-50% of the total mass of the composition, and in which the total mass of oligomeric sugar diacids derived from alginic acid, with m+m'=1 or 2, is at least 50% of the total mass of the composition. 2. The composition of oligomeric sugar diacids obtained from alginic acid according to claim 1, in which the total mass of oligomeric sugar diacids obtained from alginic acid with m+m'=1 or 2 is 60-90%, more preferably 70-90 %. 3. The composition of oligomeric sugar diacids obtained from alginic acid according to claim 2, in which the total mass of oligomeric sugar diacids obtained from alginic acid, with m+m'=1, is at least 10% of the total weight of the composition, preferably 30 -40%. 4. The composition of oligomeric sugar diacids obtained from alginic acid according to claim 1, in which the total mass of oligomeric sugar diacids obtained from alginic acid with m+m'=2 is at least 10% of the total weight of the composition, preferably 30- 50%. 5. The composition of oligomeric sugar diacids obtained from alginic acid, according to claim 1, in which the total mass of oligomeric sugar diacids obtained from alginic acid, with n=1-5, is 80-95% of the total weight of the composition. 6. The composition of oligomeric sugar diacids obtained from alginic acid, according to claim 1, in which the total mass of oligomeric sugar diacids obtained from alginic acid, with n=1-3, is 20-70% of the total weight of the composition. 7. The composition of oligomeric sugar diacids obtained from alginic acid, according to claim 1, in which the ratio of the total mass of oligomeric sugar diacids obtained from alginic acid, with n=1-3 to the total mass of oligomeric sugar diacids obtained from alginic acid, with n=4-7 ranges from 1.0 to 3.5. 8. The composition of oligomeric sugar diacids obtained from alginic acid, according to claim 7, in which the ratio of the total mass of oligomeric sugar diacids obtained from alginic acid, with n=1-3 to the total mass of oligomeric sugar diacids obtained from alginic acid, with n=4-7 ranges from 1.0 to 3.0. 9. The composition of oligomeric sugar diacids obtained from alginic acid according to claim 1, in which the total mass of guluronic acid is 1-30%. 10. The composition of oligomeric sugar diacids obtained from alginic acid, according to any one of claims 1-9, in which the mass percentage of oligomeric sugar diacids obtained from alginic acid with different degrees of polymerization is: disaccharides 5-25%, trisaccharides 15- 30%, tetrasaccharides 15-28%, pentasaccharides 10-25%, hexasaccharides 5-15%, heptasaccharides 310%, octasaccharides 2-5%, nonasaccharides 1-5%, decasaccharides 1-5%. 11. The composition of oligomeric sugar diacids obtained from alginic acid according to claim 10, in which the mass percentage of oligomeric sugar diacids obtained from alginic acid with different degrees of polymerization is: disaccharides 10-20%, trisaccharides 18-30%, tetrasaccharides 15-28%, pentasaccharides 15-20%, hexasaccharides 5-10%, heptasaccharides 3-5%, octasaccharides 2-3%, nonasaccharides 1-3%, decasaccharides 1-3%. 12. Composition of oligomeric sugar diacids obtained from alginic acid, according to any - 22 045761 from claims 1 to 11, in which the pharmaceutically acceptable salt is a sodium or potassium salt. 13. A pharmaceutical composition comprising an effective amount of an alginic acid-derived oligomeric sugar diacid composition according to any one of claims 1 to 12 and a suitable carrier, wherein the pharmaceutical composition may be a medical product. 14. Use of a composition of oligomeric sugar diacids derived from alginic acid according to any one of claims 1 to 12 in the manufacture of a medicinal product or medical product for the treatment of Alzheimer's disease, Parkinson's disease, inflammation, pain, diabetes mellitus or vascular dementia. 15. Composition of oligomeric sugar diacids obtained from alginic acid according to any one of claims 1 to 12, suitable for use as a medicine or medical product.