RU2627435C1 - Agent with antithrombotic activity - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: antithrombotic agent based on sulfated cellulose material is cellulose sulphate with a molecular weight of 116000 daltons derived from partially hydrolyzed wheat straw pulp by sulfation with chlorosulfonic acid in pyridine.

EFFECT: agent of the invention is characterized by high antithrombotic and low hemorrhagic activity.

1 tbl, 3 ex

Description

Cardiovascular disease is the leading cause of death worldwide; among them, thromboembolic complications associated with the formation of a thrombus in the cardiovascular system. The formation of a blood clot leads to stagnation of blood, changes in blood coagulation, damage to the vascular wall and changes in the concentrations of leukocytes or platelets. The invention relates to medicine and can be used to create an effective tool for the prevention and treatment of venous thrombosis of various origins in people (deep vein thrombosis, heart attacks, strokes).

The mammalian hemostatic system performs a protective function. Sequential activation of coagulation factors on phospholipid surfaces (tissue factor on cells or activated platelets), accompanied by the inclusion of positive and negative feedback mechanisms, leads to the formation of thrombin, while the anticoagulant system inhibits excessive thrombin generation, and the fibrinolysis system provides clot lysis. Violation of the balance of procoagulant and anticoagulant mechanisms leads to thrombosis or bleeding. With thrombosis, blood clots appear in the vessels, containing mainly fibrin platelets (arterial thrombosis) or fibrin erythrocytes (venous thrombosis) [1].

Currently, for the prevention and treatment of thrombosis, along with other drugs, anticoagulants are also used, which also have antithrombotic activity. However, all modern anticoagulants, regardless of the mechanism of action (direct inhibitors of thrombin or activated factor X activity and antithrombin activators are heparin preparations, in structure are sulfated polysaccharides), have a number of side effects, the main of which is the ability to cause bleeding. Search for new compounds in addition to heparin preparations (unfractionated heparin with the same antithrombin-mediated inhibitory activity against thrombin and factor Xa; low molecular weight heparins with less than that of unfractionated heparin, antithrombin-mediated inhibitory activity against thrombin; synthetic pentasaccharide - fondaparine antithrombin-mediated inhibitory activity against factor Xa, without activity against thrombin), but with less hemorrhagic activity Is carried out in a number of sulfated polysaccharides of plant, animal, for example amongst marine invertebrates and algae, as well as among terrestrial plant tissues [2, 3]. However, the structural heterogeneity of polysaccharides from algae makes it difficult to obtain compounds with programmable constant high activity. Therefore, some authors choose linear polysaccharides isolated from land plants as a substance for subsequent chemical modification of polysaccharides [4]. For example, cellulose is a polysaccharide whose macromolecule is composed of repeating units - β-D-glucopyranose residues with the general formula (C ₆ H ₁₀ O ₅ ) n, which is the most common organic compound that is isolated from herbaceous and tree-like plants.

The closest in essence and purpose to the present invention is an antithrombotic agent - cellulose sulfate with a molecular weight of 80,000 Da [5], obtained from partially hydrolyzed fir cellulose by sulfation with a complex of pyridine and chlorosulfonic acid, cellulose hydrolysis was carried out according to the method described in RF patent No. 2203995 [ 6].

The disadvantage of this invention is the low antithrombotic activity of the sulfated polysaccharide. The disadvantage of the invention is due to the type of plant material used for the separation of cellulose, and the insufficient molecular weight of cellulose sulfate (80,000 Da), which is associated with harsh conditions for partial hydrolysis of the original cellulose.

It is known that the anticoagulant activity of polysaccharides depends on a number of structural parameters — the degree of sulfation, the location of functionally active groups, the charge density, and the molecular weight of polysaccharide sulfates, with an increase in which the anticoagulant activity of polysaccharides obtained from plant materials sometimes increases [7].

The objective of the invention is the expansion of the range of antithrombotic agents based on plant polysaccharide with high antithrombotic activity.

EFFECT: obtained product with high antithrombotic activity based on chemically modified wheat straw cellulose.

The technical result is achieved in that the antithrombotic agent based on sulfated cellulosic material is cellulose sulfate with a molecular weight of 116,000 Da, obtained from partially hydrolyzed cellulose of wheat straw by sulfation with chlorosulfonic acid in pyridine.

In contrast to the prototype in the present invention, upon receipt of an antithrombotic agent, partial hydrolysis of wheat straw pulp is carried out at a temperature of 100 ° C for 1 h, in the prototype, more stringent conditions: temperature 120 ° C, duration of hydrolysis 2 hours

Sulfation of partially hydrolyzed cellulose of wheat straw to obtain an antithrombotic agent was carried out according to the procedure described in RF patent No. 2426746 [8], as follows.

Cellulose isolated from wheat straw was previously subjected to mild hydrolytic action of an aqueous solution of the composition: 6.4% hydrogen peroxide; 23.6% acetic acid, 2% sulfuric acid, with a water module equal to 10; at a temperature of 100 ° C for 1 h. Then, the cellulose was subjected to preliminary swelling in a pyridine medium for at least 12 h. The cellulose thus prepared at 10-15 ° C was added to the complex of pyridine and chlorosulfonic acid. Sulfation was carried out at 80-85 ° C for 2-2.5 hours. Wheat straw cellulose sodium salt was isolated from the reaction mass by decantation of pyridine, neutralization of the viscous residue washed with ice water with a 6% aqueous sodium hydroxide solution to pH 8-9 by decantation of an aqueous solution of cellulose sulfate sodium gel gel, dissolving the salt in water and planting a 3-fold volume of ethanol. The result was a water-soluble sodium salt of cellulose sulfate with a basic substance content of 98% (mass.), Sulfur content (ω _s ) of 14.1% (mass.); degree of substitution (Cs) 1.3 and a molecular weight MM 116,000 Yes. It was established by ¹³ C NMR that all the hydroxyl groups of cellulose at the C ₆ carbon atom are replaced by sulfate groups, and at the C ₂ atom, they are partially replaced by sulfate groups.

The degree of substitution in the samples was found by the equation: CЗs = 162⋅ω _s / 3200-ωs⋅103, where ω _s is the sulfur content,% [9]. Sulfur content was determined on a flash EA-1112 elemental analyzer (Thermo Quest Italia).

¹³ C NMR spectra of the initial and sulfated cellulose of wheat straw were recorded at 25 ° C using a 600 MHz Bruker Avance III spectrometer in D ₂ O with reference to solvent deuterium resonance.

Molecular mass distribution (MMP) was studied on an Agilent 1200 chromatograph with a PL aguagel column — OH 40, 300–7.5; refactometric detector. As a solvent and an eluent, an aqueous solution of 0.1 M LiNO _{3 was used} .

The specific anticoagulant activity of the obtained substance of cellulose sulfate (SC) was evaluated by the time of coagulation of human blood plasma in coagulological tests of activated partial thromboplastin time (APTT) and using the ReaKlot-Heparin test (NPO Renam, Moscow). To calculate the antithrombin (aIIa) and antifactor Xa (aXa) activities, calibration curves of the International Standard for Unfractionated Heparin (UFH) were used.

The high antithrombin activity of the SC made it possible to evaluate antithrombotic activity. The study was carried out using laboratory animals (rats), which were obtained from the nursery RAMS "Pillar 2". All experiments were carried out in compliance with the "Rules for the work using experimental animals" (Order of the USSR Ministry of Health of 12.08.1977 No. 755 "On measures to further improve the organizational forms of work using experimental animals) and the" Rules of laboratory practice "(annex to the Order of the Ministry of Health and Social Development Russia of August 23, 2010 N 708n). As a comparison drug used the International standard UFH. To assess antithrombotic activity, a model of venous thrombosis in rats according to S. Wessler was used [10]. With an increase in the dose of SC and UFH, antithrombotic activity increased. To achieve 50%, inhibition of SC thrombus growth was required 2 times more than UFH.

The invention is illustrated by the following examples.

Example 1. Determination of antithrombin activity of the substance SC. To a mixture of plasma and APTT reagent (0.15 ml) (NPO Renam) was added a solution of SC or NFH (International Standard) at final concentrations of 0.0005-0.0025 μg / ml. After 3 minutes of incubation at 37 ° C, 0.02 ml of a 0.025 M CaCl ₂ solution was added and the time (sec) for the appearance of the fibrin clot was recorded on a Minilab 701-M coagulometer. The effect of different concentrations of SC on the coagulation time of human plasma in the APTT test is shown. Comparing the readings of the SC concentration – plasma clotting curves in 15 independent determinations with the UFH calibration curves, the antithrombin (aIIa) activity of the SC was determined [10]; aIIa SC activity was 93.8 ± 1.0 U / mg.

Example 2. Determination of antifactor Xa activity of the substance SC. To a mixture of plasma and a solution of factor Xa (0.16 ml; test ReaKlot-Heparin, NPO Renam) was added SC or NFH (International Standard NFH) at final concentrations of 0.0002-0.001 μg / ml. After 2 min incubation at 37 ° C, 0.02 ml of a 0.035 M CaCl ₂ solution was added and the time (sec) of the appearance of the fibrin clot was recorded on a Minilab 701-M coagulometer. The influence of SC on the coagulation time of human plasma in the ReaKlot-Heparin test is shown. Comparing the readings of the SC concentration-time plasma clotting curves in 15 independent determinations with the UFH calibration curves, the antifactor Xa (aXa) of the SC activity was determined [10]; aXa activity of SC was 9.9 ± 0.6 U / mg.

Example 3. Antithrombotic activity of SC was evaluated by modeling venous thrombosis according to Wessler S. et al. [11] in 55 Wistar male rats weighing 180-350 g. For anesthesia, rats were administered intraperitoneally at a dose of 60 mg / kg. SC solutions in doses of 1, 3, and 5 mg / kg were injected into the left jugular vein (the comparison drug UFH was administered in doses of 1 and 1.5 mg / kg), and after 15 min, human-activated glass serum. Then, a section of the opposite vein, which was not used for the administration of anticoagulants, was ligated with a thread, after 15 minutes, the bandaged section of the vein was cut out and the contents were extracted into a small container. The effectiveness of antithrombotic activity was evaluated: 1) in points from 0 (no thrombus) to 4 (one large thrombus) in the form of a thrombus extracted from a ligated section of a vein; 2) by protein concentration in the thrombus homogenate, measured according to Lowry; the protein content in the test sample was determined by a calibration curve constructed with a solution of human fibrinogen (Sigma Aldrich). With an increase in the dose, SC decreased: the form of blood clots from 2.00 ± 0.49 to 0.10 ± 0.10 points and the protein concentration in blood clot homogenates from 1.19 ± 0.17 to 0.02 ± 0.02 mg / ml (tab. 1). Significant differences in the form of blood clots were noted between the indications with the introduction of saline and SC, starting with a dose of 3 mg / kg.

The advantage of the proposed tool in comparison with the prototype is a large 10 U / mg antithrombin activity and a 2-fold lower Ax activity, which can lead to less hemorrhagic activity when the rats are given cellulose sulfate intravenously.

Bibliography

1. Nakamura M., Yamada N., Ito M. Current management of venous thromboembolism in Japan: Current epidemiology and advances in anticoagulant therapy. // J Cardiol., 2015 .-- 66 (6). - 451-459.

P.A. Perspective on the use of sulfated polysaccharides from marine organisms as a source of new antithrombotic drugs. // Mar Drugs. - 2015. - 13(5). - 2770-2784.2.

PA Perspective on the use of sulfated polysaccharides from marine organisms as a source of new antithrombotic drugs. // Mar Drugs. - 2015 .-- 13 (5). - 2770-2784.

K., Kovac Besivic E., Njksic H. et al. Anticoagulant activity of some Artemisia dracunculus leaf extracts // Bosn J Basic Med Sci. - 2015. - 15(2). - 9-14.3.

K., Kovac Besivic E., Njksic H. et al. Anticoagulant activity of some Artemisia dracunculus leaf extracts // Bosn J Basic Med Sci. - 2015 .-- 15 (2). - 9-14.

4. Ehmann H. M., Mohan T., Koshanskaya M. et al. Design of anticoagulant surfaces based on cellulose nanocrystals // Chem. Commun. (Camb.). 2014.- 50 (86). - P. 13070-13072.

5. RF patent No. 2571555. Antithrombotic agent from Siberian fir cellulose // Kuznetsova S.A., Drozd N.N., Savchik E.Yu., Miftakhova N.T., Vasilyeva N.Yu., Makarov V.A., Kuznetsov B.N. Publ. 12/20/2015.

6. RF patent No. 2203995. A method of producing microcrystalline cellulose // Danilov V.G., Yatsenkova O.V., Kuznetsova S.A., Kuznetsov B.N. Publ. 05/10/2003.

7. Arata P., Quintana I., Canelon D. et al. Chemical structure and anticoagulant activity of highly pyruvylated sulfated galactans from tropical green seaweeds of the order Bryopsidales // Carbohydr Polym. 2015. - 122. - P. 376-386.

8. RF patent No. 2426746. A method of producing sulfated cellulose // Vasilyeva N.Yu., Kuznetsova S.A., Kuznetsov B.N., Skvortsova G.P., Danilov V.G. Publ. 08/20/2011.

9. Torlopov M.A. The production of powder materials by the destruction of cellulose by Lewis acids and their modification. Sulphation of powder materials obtained by the destruction of cellulose by Lewis acids // M.A. Torlopov, S.V. Frolova // Chemistry of plant raw materials. - 2008. - No. 3. - C 63-67.

10. Makarov V.A., Spasov A.A., Plotnikov M.B., Chernysheva G.A., Blozerskaya G.M., Vasilieva T.M., Drozd N.N. and others // Methodological guidelines for the study of pharmacological substances that affect hemostasis. S. 453-479 // Guidelines for preclinical studies of drugs. Part One // Ed. A.N. Mironova.- M .: Grif and K. 2012 .-- 944 p.

11. Wessler S., Reimer S.M., Sheps M.S. Biologic assay of a thrombosis-inducing activity in human serum // J. Appl Physiol 1959. - V.14. - No. 1. - P. 943-946.

Claims (1)

An antithrombotic agent based on sulfated cellulosic material, characterized in that it is a cellulose sulfate with a molecular weight of 116,000 Da, obtained from partially hydrolyzed cellulose of wheat straw by sulfation of chlorosulfonic acid in pyridine.