RU2240123C1 - Exogenous biologically active conjugating substances - Google Patents

Abstract

FIELD: medicine, medicinal biochemistry.

SUBSTANCE: invention relates to applying polyuronic acids or their salts as agents used in substitution therapy of diseases associated with disturbance of metabolic processes, in particular, conjugation reactions with glucuronic acid (renal coma, hepatocellular insufficiency, hepatitis, jaundices and others). Agent for substitution therapy comprising uronic acids involves polygalacturonic acid with esterification degree 40.4% (beet pectin) or derivative of polymannuronic and polyguluronic acid sodium salts (sodium alginate) as uronic acids. Agent promotes to effective protection of patients with disturbance of metabolic processes.

EFFECT: valuable medicinal and biochemical properties of agents.

5 tbl, 4 ex

Description

The invention relates to medicine and relates to the use of polyuronid acids or their salts as a means of substitution therapy for diseases associated with metabolic disorders, in particular conjugation reactions with glucuronic acid. Such diseases include hemolytic disease, hepatic coma, hepatic cell failure, hepatitis, jaundice, etc.

A significant place in the metabolism of various substances in the body belongs to conjugation mechanisms. Various substances are conjugated with various endogenous compounds (glucuronic, sulfuric acid, etc.). Conjugation with glucuronic acid is the most important conjugation mechanism in humans and includes two main stages: biosynthesis of the coenzyme complex of glucuronic acid and transfer of the glucuronide moiety to the inactivated substance from this complex. An example of such a conjugation is the conjugation of bilirubin:

Уридиндифосфат-α-D-глюкоза (УДФГ)

Uridinediphosphate-α-D-glucose (UDFG)

Uridinediphosphate-α-D-glucoronic acid

Bilirubin glucuronide (p-configuration) (Vasilenko Yu.K. Biological chemistry. - M.: VSh, 1978. - S.370-371; Balakhovsky SD, Balakhovsky I.S. Methods of the chemical analysis of blood. - M. : Medgiz, 1953.- P.588).

The important physiological role of the formation of glucuronides (β-D-glucuronic acid glycosides) is due to the fact that this reaction is one of the main conjugation reactions leading to the neutralization and elimination of drug and toxic substances and some metabolites. These include many normal metabolites (bilirubin, steroids, thyroxine, etc.) and foreign substances (metabolic products of the intestinal microflora, drugs, chemical environmental pollution). The pathogenesis of hemolytic disease of the newborn is based on the insufficiency of glucuronyl transferase, which catalyzes the formation of bilirubin glucuronide, i.e. the form of bilirubin that is excreted from the body; as a result of this enzyme, bilirubin accumulates in the blood and tissues. The pathogenesis of symptoms of hepatic cell failure and hepatic coma is associated with poisoning of the body with toxic substances due to a violation of the mechanism of neutralization reactions in the liver, in particular reactions of the formation of glucuronides (Big Medical Encyclopedia. - M .: Sov. Encyclopedia, 1976. - V. 4. - Ed. .3. - S.325).

β-D-Glucuronic acid is an endogenous substance, i.e. substance formed in the human body as a result of enzymatic oxidation of glucose.

Of the drugs containing glucuronic acid, only one drug is known - honsuridum (Chonsuridum) - a drug obtained from cattle trachea (Mashkovsky M.D. Medicines: In 2 volumes - T.2. - 10th ed., ster. - M .: Medicine, 1986. - P.156). The active substance of honsuride is chondroitinsulfuric acid, built from the molecules of N-acetylgalactosamine-6-sulfate and glucuronic acid. This tool is close to the claimed and selected for the prototype. The disadvantages of this drug are: 1) the impossibility of its use for conjugation reactions that contribute to the neutralization and release of various metabolites from the body, because in chondroitin sulfate, glucuronic acid is bound to N-acetylgalactosamine-6-sulfate with a very strong β (1 → 4) -glycoside bond, and to participate in the conjugation reaction, glucuronic acid must be bound in UDFGK (complex with macroergic bond); 2) the inability to use honsurida inside, because He along with hyaluronic acid is involved in the construction of the main substance of connective tissue.

By their chemical structure, polyuronides, such as pectins and alginates, are related compounds of glucuronic acid. Pectins are biopolymers consisting of D-galacturonic acid residues interconnected by α (1 → 4) -glycoside bonds. Polygalacturonic acid is partially esterified with methanol, which contributes to the dissolution of pectin in water (Komissarenko S.N., Spiridonov V.N. Pectins - their properties and application. Review. / Growth resources, 1998. - V. 34. - Issue 1. - S.111-119). Alginic acids are heteropolyuronides, the molecules of which are built from the residues of β-D-mannuronic and α-L-guluronic acids linked in linear chains by 1-4-glycosidic bonds. Because Since alginic acids are slightly soluble in water, and their sodium salts are easily soluble in water, the latter are most often used (Chemical Encyclopedia. - M .: Scientific Publishing House “Big Russian Encyclopedia”, 1998. - T.1. - C .192-193). The chemical formulas of the polyuronide monomers (Tyukavkina N.A., Baukov Yu.I. Bioorganic chemistry. - M .: Medicine, 1985. - P.352-354, 375-376) are given below:

As can be seen from the above chemical formulas of hexuronic acids, glucuronic and galacturonic acids are epimers for the hydroxyl group at C ₄ ; glucuronic and mannuronic acids are epimers in the hydroxyl group at C ₂ , and guluronic and mannuronic acids are epimers in the hydroxyl group at C ₅ .

The similar structure of the hexuronic acids mentioned above suggested the possibility of replacing glucuronic acid in conjugation reactions with other uronic acids or their salts of exogenous origin. As conjugating substances, we selected beet pectin (SP) with an degree of polyester esterification of polygalacturonic acid of 40.4% and sodium alginate (AN) isolated from saccharose kelp.

The purpose of the invention is the study of the possibility of using plant polyuronides as conjugating substances instead of glucuronic acid in violation of biosynthesis or metabolism of the latter in the human body.

Various factors are known that impede the course of conjugation reactions with glucuronic acid: a) heavy metal ions block glucuronic acid, glucose-1-phosphate (the starting material for the synthesis of UDFG). Heavy metal compounds block the functional groups (carboxyl, sulfhydryl, phosphoric, amine, etc.) of biomolecules, forming compounds lacking many physicochemical and biological properties, thereby violating the metabolism in general and glucuronide conjugation, in particular (Emergency care for acute poisoning. Handbook of Toxicology / Edited by S.N. Golikov. - M .: Medicine, 1978. - S. 214-215); b) inhibition of the enzyme UDF-glucuronyl transferase and other enzymes by heavy metal ions, parachloromercuriebenzoate (Big Medical Encyclopedia), which inhibits the transfer of the glucuronide part from UDFGK to an inactivated substance and causes poisoning of the body with toxic substances due to the accumulation of the latter in blood and tissues; c) genetic defects of enzymes (Human Biochemistry. / Murray R., Grenner D., Meyes P., Rodwell V. - M .: Mir, 1993. - In 2 volumes - V.1. - 382 p.) .

This goal is achieved by first creating a model of metabolic disturbance by daily oral administration of white rats male solution of lead (II) acetate at a dose of 45 mg / kg for a week, and then a solution of SP or AN is administered orally daily for three months. at a dose of 500 mg / kg. In animals, the content of hexuronic acids and bilirubin (a normal metabolite for the human body) in blood serum and urine was studied throughout the experiment. In order to identify the effect of exogenous polyuronides on the metabolism of xenobiotics, in particular aspirin, animals after a weekly administration of lead (II) acetate and monthly administration of polysaccharides daily are administered orally at a dose of 30 mg / kg in suspension as a suspension with 5 ml of water daily for a week after eating. A group of intact animals served as a comparison.

The content of hexuronic acids in biological material was determined after acid hydrolysis of a biosubstrate by reaction with carbazole in a sulfuric acid medium by measuring the light absorption of a pink-raspberry colored reaction product at a wavelength of 530 nm (Research methods in occupational pathology (biochemical). Manual for doctors. / Arkhipova O .G., Shatskaya M.M., Semenova L.S. et al. / Edited by O.G. Arkhipova. - M .: Medicine, 1988. - 203 p.).

Detection of bilirubin in urine was carried out according to the Rosin test, based on the formation of bilirubin under the action of biliverdin iodine, colored green (Stroev EA, Makarova VG Workshop on biological chemistry. - M .: VS, 1986. - C .188).

The quantitative content of direct bilirubin in blood serum was determined by a dilution method based on the interaction of a diazoreakt with bilirubin in the most diluted blood serum (Balakhovsky S.D., Balakhovsky I.S. Methods of a chemical analysis of blood. - M.: Medgiz, 1953. - P. 586).

The content of oxybenzoic acid (aspirin) in biological fluids was determined by the acid-base titration method (State Pharmacopoeia of the USSR. - M .: Medicine, 1968. - X ed. - Art. 3).

Previously (before creating a model of metabolic disturbance), the effect of SP and AN on the level of hexuronic acids in various biological systems and organs of animals was studied. The results are shown in table 1 (see the end of the description).

The data presented in table 1 indicate that as a result of oral administration of SP and AN to animals, the level of hexuronic acids in all biosubstrates increases: it rises most rapidly in blood serum and soft tissues, and slowly in bone tissue. An increase in the content of hexuronic acids, especially in blood serum, in the liver suggests the possibility of the participation of introduced polyuronides in metabolic processes.

When determining the presence of bilirubin in the urine using the Rosin test in each group of animals throughout the study period, the reaction to bilirubin was negative, which indicated the normal course of metabolic processes in the liver.

To create a model of metabolic disturbance, white male Wistar rats weighing 170-220 g weighing 45 mg / kg body weight (in terms of lead) were orally administered daily for 7 days, then lead to a solution of SP or AN in doses of 500 mg / kg body weight daily for 12 weeks. The contents of hexuronic acids (in terms of glucuronic acid) and bilirubin were determined periodically in blood and urine. The results are shown in table 2.

The results shown in table 2 show that after administration of a solution of lead (II) acetate in the blood and urine, the bilirubin content increases in the blood, while the content of hexuronic acids in the blood decreases, and in the urine it increases. Apparently, this can be explained by the fact that under the influence of lead, transferases are blocked, catalyzing the transfer of the glucuronide part from UDFGK to bilirubin, i.e. the introduction of lead disrupts the synthesis of glucuronide-bilirubin, therefore, in both blood and urine, the content of unbound bilirubin increases. For the same reason, the content of glucuronic acid in the form of uridine coenzymes in urine also increases. The decrease in the content of glucuronic acid in the blood is probably due to the binding by lead ions of the carboxylic acid groups of uronic acid or the phosphate groups of glucose-1-phosphate at the stage of UDFG synthesis.

Against the background of lead pathology, the subsequent long-term administration of polyuronide preparations leads to the disappearance of bilirubin in the urine (after 8 weeks of taking polysaccharides) and a decrease in its content in the blood to normal values (after 4 weeks of administration). In this case, the content of hexuronic acids in blood serum and urine is normalized after a 4-week intake of polyuronides. The beneficial effect of polyuronide drugs on metabolism, in particular bilirubin, can apparently be explained by the involvement of monomer fragments of the introduced polyuronide drugs in the conjugation process.

To establish the relationship between the content of hexuronic acids and direct bilirubin in the blood serum, as well as to establish the nature of the dependence and quantitatively express the reliability of the relationship, the correlation coefficient (r) between the two indicated characteristics was calculated by a known method (Asatiani BC New methods of biochemical photometry. - M .: Nauka, 1965 .-- S. 507-510). The calculation of the correlation coefficient between the content of hexuronic acids and direct bilirubin in the blood serum after the introduction of polyuronide preparations is shown in table 3.

The data in table 3 show that there is a relationship between the content of hexuronic acids after the introduction of polyuronides and direct bilirubin in the blood serum. A negative value of the correlation coefficient indicates an inverse correlation between the considered features. The coefficient values, very close to unity, indicate a high probability of a relationship between the indicators in both cases of the use of polysaccharides.

The established degrees of probability show that there is a statistically significant inverse relationship between the content of hexuronic acids after the administration of polyuronide preparations and the content of direct bilirubin. This confirms the correctness of the assumption that a decrease in the bilirubin content in blood serum was a consequence of an increase in the content of uronic acids due to the introduction of polyuronide preparations.

Along with normal metabolites, which include bilirubin, an attempt has been made to study the relationship between the content of hexuronic acids after taking polyuronides and the content of any xenobiotic metabolized in the body by glucuronide conjugation. As such a xenobiotic, aspirin was chosen. In the liver, it forms a conjugate with glucuronic acid (Tyukavkina N.A., Baukov Yu.I. Bioorganic chemistry. - M .: Medicine, 1985. - P.376).

First, a model of metabolic disturbance was created by oral administration of lead (II) acetate solution to white males for one week (as in the case described above). Then, against the background of lead pathology, one group of animals was orally administered weekly daily (after eating) with a probe, aspirin powder at a rate of 30 mg / kg body weight in the form of a suspension in 5 ml of water. After a weekly lead injection, the other two groups of animals were given oral solutions of SP or AN in doses of 500 mg / kg body weight daily for 4 weeks and only then aspirin was introduced (similar to the first group of animals). In the urine of animals, the content of oxybenzoic acid was determined by the acid-base titration method (State Pharmacopoeia of the USSR. - M .: Medicine, 1968. - Xth ed. - Art. 3). The results of the quantitative determination of the content of oxybenzoic acid in the urine of animals are presented in table 4.

The data in table 4 show that as a result of the administration of aspirin against the background of lead pathology, when metabolic processes are disturbed, the content of free, unconjugated, hydroxybenzoic acid in the urine increases, because, apparently, the uronide component of the transferase enzyme is inhibited by lead ions it is not transferred from UDFGK to an inactivable substance (salicylates), which leads to an increased content of hexuronic acids in the urine. Presumably, glucuronic acid is blocked by lead ions and cannot participate in the conjugation of metabolites. The introduction of aspirin against the background of lead pathology and prolonged intake of polyuronides indicates the normalization of metabolic processes: with a decrease in the content of salicylates, the content of hexuronic acids in the urine also decreases. To determine the presence of a relationship between two signs (the content of hexuronic acids and salicylates in the urine after administration of aspirin against a background of lead and polyuronides), a correlation coefficient was calculated. The calculation results are shown in table 5.

The data in table 5 show that between the content of hexuronic acids (after the introduction of polyuronides) and the content of salicylates in the urine there is a connection. A positive value of the correlation coefficient indicates a direct correlation between the considered indicators. In the case of the introduction of AN, the correlation coefficient is practically 1 (0.992), which indicates a high degree of probability of the presence of a relationship between the indicators. This confirms the correctness of the assumption that a decrease in the content of oxybenzoic acid (salicylates) in the urine was a result of a decrease in the content of uronic acids due to the administration of polysaccharides.

Along with the endogenous conjugating substance - glucuronic acid - after taking polysaccharide preparations, other uronic acids (galacturonic, mannuronic, guluronic) appear in biological organs and systems. It can be assumed that either these other uronic acids play the same role of conjugating substances as glucuronic acid, or exogenous uronic acids are converted to glucuronic acid, as is observed during enzymatic transformations occurring in plants (Kretovich V.L. Plant biochemistry .-- M .: VSH, 1980. - S.193):

The second assumption is less likely due to the possible lack of appropriate enzymes in the human body.

Along with the described pronounced biological effect, the use of SP and AN is harmless to humans, because both polysaccharides are food components and are biocompatible with the body.

The study of the “acute” toxicity of SP and AN was carried out by the Kerber method (KK Sidorov. Methods for the determination of acute toxicity and chemical hazard (toxicometry). - M .: Medicine, 1970) on 20 white male rats weighing 180-220 g by oral administration. the introduction of 5 ml of a solution of SP or AN at a dose of 3500 mg / kg body weight. After a single injection during the observation period (within 14 days), no deaths were observed, which allowed us to determine LD ₅₀ when administered orally> 3500 mg / kg (for both polysaccharides). According to the classification of toxic substances (Sidorov K.K., 1970), both polysaccharides studied belong to the group of low toxic substances.

The inventive method is illustrated by the following examples.

Example 1

A portion of lead (II) acetate weighing 3.5313 g is dissolved in 100 ml of water by stirring the flask with the solution on a magnetic stirrer, then the solution is quantitatively transferred into a volumetric flask with a capacity of 250 ml. To suppress the hydrolysis of lead (II) ions and acetate ions, a few drops of a 0.1 mol / L acetic acid solution are added to the resulting solution until the turbidity disappears. Then the solution was adjusted with water to the mark. 1 ml of the prepared solution of lead (II) acetate contains 9 mg of lead (II) ions.

The experiments were performed on 60 sexually mature white male Wistar rats weighing 180-220 g, divided into 6 groups of 10 animals each. The first group of animals - intact - served as a comparison. The remaining groups (II-VI) of animals daily for 7 days orally (using a probe) were injected with a solution of lead acetate (II) with a volume of 1 ml at a dose of 45 mg / kg (in terms of lead). Group II animals served as control. Then, III-VI groups of animals were orally administered a solution of beet pectin (5 ml) daily at a dose of 500 mg / kg for 2 weeks (Group III), 4 weeks (Group IV), 8 weeks (Group V) and 12 weeks (VI Group).

To prepare a pectin solution, a 10 g sample of weighed mass was weighed, transferred to a 1 L conical flask, and 500 ml of water was added, i.e. prepared a 2% solution of SP. For swelling and dissolution of pectin in water, the solution was left for a day at room temperature.

All animals during the experiment were on a standard diet. In animals of all groups, the content of hexuronic acids (in terms of glucuronic acid) in blood serum and urine was studied using the carbazole method (Research methods in occupational pathology (biochemical) ...), the presence of direct bilirubin in the urine by Rozin's test (E.A. Stroev, Makarova V.G. et al.) And the content of direct bilirubin in blood serum (Balakhovsky S.D. et al.).

To obtain urine, 5 ml of water was administered intragastrically to rats using a probe, then the animals were sedimented for a day in a urine collector. The collected urine was filtered through a paper filter before analysis.

For blood sampling, animals were decapitated. The collected blood was defended in the refrigerator to separate the serum.

The results of biochemical analysis of biological fluids are shown in table 2. The probability of differences in results with respect to the control does not exceed 0.05, which is within normal limits and indicates the statistical reliability of the data.

Example 2

Carried out analogously to example 1, but instead of a 2% solution of SP use a 2% solution of sodium alginate (AN).

To prepare a 2% AN solution, a 10 g sample of AN is weighed, placed in a 1 L conical flask, 500 ml of water are added, and the solution is stirred until the AN is dissolved.

The results of a biochemical analysis of blood serum and urine for the content of hexuronic acids and direct bilirubin are shown in table 2. The results are statistically significant.

Example 3

Solutions of lead (II) acetate and SP were prepared as described in example 1.

The experiments were performed on 50 Wistar male rats, divided into 5 groups of 10 animals each. The first group is intact animals. For the remaining groups (II-V, except III), the animals were orally administered weekly with a solution of lead (II) acetate 1 ml daily at a dose of 45 mg / kg (in terms of lead) for a week. Then, the IV group (against the background of lead pathology; control) and the III group of animals were orally administered with aspirin powder daily at a dose of 30 mg / kg body weight in the form of a suspension in 5 ml of water daily for a week after a meal. Next, the 5th group of animals (against the background of lead pathology) was orally administered daily with a 2% 5 ml SP solution at a dose of 500 mg / kg for 4 weeks and then a suspension of 5 ml aspirin at a dose of 30 mg / kg for 1 week daily after meal.

In animals of all groups, at the end of the experiment, and in the V group, the urinary content of hexuronic acids (in terms of glucuronic acid) was studied daily within 4 days after the administration of aspirin using the carbazole method (Research methods in occupational pathology (biochemical) ...) and hydroxybenzoic acid by acid-base titration (State Pharmacopoeia of the USSR ...).

The results of biochemical studies are statistically significant and are shown in table 4.

Example 4

Carried out analogously to example 3, but instead of a 2% solution of SP use a 2% solution of AN (its preparation is described in example 2).

The results of biochemical studies are shown in table 4.

Thus, the acidic polysaccharides offered as conjugating substances provide the following beneficial effect:

1) with the introduction into the body of intact animals acidic polysaccharides (SP, AN), the content of hexuronic acids in biosystems increases; in soft tissues, an increase occurs earlier than in bone tissue. An increase in the content of uronic acids in the blood serum, liver, and urine makes it possible to suggest the participation of the introduced polyuronides together with glucuronic acid in metabolic processes, in particular glucuronide conjugation.

2) As a result of the weekly administration of lead (II) acetate, metabolic processes are disturbed: the content of hexuronic acids in the blood serum decreases by 1.7 times, and in the urine - increases by 3 times; the content of free bilirubin (a metabolite normal for the body) in the blood serum increases by 2.9 times, and bilirubin also appears in the urine. Perhaps this is due to the fact that lead (II) ions block the functional groups of both glucose-1-phosphate, UDP-glucuronic acid, and the transferase enzyme, which catalyzes the transfer of the uronide moiety from UDFGK to bilirubin, as a result of which glucuronide conjugate does not form β-bilirubin and free bilirubin are found in urine and blood. In part, due to blocking by lead (II) ions, UDP-glucuronic acid cannot conjugate bilirubin. The subsequent long-term administration of SP and AN normalizes metabolic processes: after 4 weeks of taking polysaccharides in blood serum and urine, the content of hexuronic acids normalizes, which leads to normalization of the bilirubin content in the blood, and after 8 weeks of taking polyuronide preparations, bilirubin is not detected in the urine. The beneficial effect of polyuronides on the metabolism of bilirubin, usually excreted in the form of conjugates with glucuronic acid, can apparently be explained by the formation of conjugates of bilirubin with acidic polysaccharides introduced into the body with a lack of glucuronic acid.

3) An inverse relationship has been established between the content of hexuronic acids, administered in the form of SP and AN, and bilirubin in blood serum. Correlation coefficients for SP and AN, respectively, are -0.984 and -0.996, which indicates a high degree of probability of a relationship between the content of hexuronic acids and bilirubin in the blood.

4) The beneficial effect of SP and AN on the metabolism of not only normal metabolites, but also on the metabolism of xenobiotics, in particular aspirin, has been established. The administration of aspirin to animals against the background of lead pathology contributes to an increase in urine of unconjugated oxybenzoic acid and glucuronic acid in comparison with the non-pathological model by 1.4 and 3.7 times, respectively. If aspirin is administered after a weekly intake of lead and a 4-week intake of polysaccharide preparations, then metabolic processes are normal. Consequently, polysaccharide preparations are included instead of glucuronic acid in the conjugation cycle with aspirin, while both urinary hexuronic acids and aspirin are reduced in the urine.

5) A direct relationship has been established between the content of hexuronic acids, introduced in the form of SP and AN, and the content of oxybenzoic acid (aspirin). Correlation coefficients for SP and AN, respectively, are +0.968 and +0.992, which proves with a high degree of probability the presence of a direct correlation between these signs.

6) Acid polysaccharides (SP, AN) are sparingly soluble in water and therefore can be introduced into the body in the form of solutions, incl. and injectable. Solutions with antimicrobial activity are stable (Pecto. Natural bactericidal enterosorbent. / Pogorelskaya L.V., Tryakina I.P., Bunov S.V. Methodical recommendations. - M., 2000. - 16 p.).

7) Being components of food products (SP is obtained from squeezed sugar beets, AN - from thalli of seaweed) and being biocompatible with the human body, SP and AN are characterized by LD ₅₀ > 3500 mg / kg when administered orally.

Claims (1)

A replacement therapy agent containing uronic acids, characterized in that it contains polygalacturonic acid with an esterification degree of 40.4% (beet pectin) or a derivative of the sodium salts of polymannuronic and polyguluronic acids (sodium alginate) as uronic acids.