RU2571555C1 - Antithrombotic agent of siberian fir cellulose - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: antithrombotic agent of sulphated cellulose material represents cellulose sulphate produced of partially hydrolysed wood substance of Siberian fir by sulphating by means of a complex of pyridine and chlorosulphonic acid.

EFFECT: agent is effective to prevent and treat thrombotic conditions and based on chemically modified compounds recovered from natural raw material.

2 dwg, 2 tbl, 4 ex

Description

The invention relates to the field of the pharmaceutical industry and medicine and can be used to create an effective tool for the prevention and treatment of thrombotic conditions in humans (heart attacks, strokes of various localization).

In modern medical practice, for the prevention and treatment of thrombosis, they also use antithrombotic (ATB) preparations of sulfated polysaccharide (glycosaminoglycan), an anticoagulant of direct action of heparin. Heparin-like polymers and sulfated polysaccharides are found in both marine invertebrate tissues and algae.

Styela plicata Santos J. et al. [1] isolated heparin from the marine invertebrate ascidia and determined the structural units: ΔUA (2SO ₄ ) -1 → 4-β-D-GlcN (SO ₄ ) (47.5%), ΔUA (2SO ₄ ) -1 → 4-β-D-GlcN (SO ₄ ) (6SO ₄ ) (38.3%), ΔUA (2SO ₄ ) -1 → 4-β-D-GlcN (SO ₄ ) (3SO ₄ ) (6SO ₄ ) (2.8%), ΔUA (2SO ₄ ) -1 → 4-β-D-GlcN (SO ₄ ) (3SO ₄ ) (8.0%). Antithrombin (aIIa) activity of heparin invertebrates, measured in the activated partial thromboplastin time test (APTT), was 18 U / mg, activity against factor Xa (aXa activity) did not exceed 2 U / mg. Therefore, in a model of venous thrombosis in rats, S.plicata heparin inhibited by 80% the increase in thrombus size by a 10-fold higher dose than was required for 100% inhibition upon administration of unfractionated heparin (UFH).

In 2012, Chen S. et al. [2] showed that the fucan polysaccharide isolated from the sea cucumber Isostichopus badionotus with a repeating tetrasaccharide link in the structure [→ 3Fuc (2c, 4S) α1 → 3Fuc (2 S) α1 → 3Fuc (2S ) α1 → 3Fuc α1 →] n S) α1 → 3Fuc (2S) α1 → 3Fuc α1 →] n, has high anticoagulant (AK, with potentiation of antithrombin activity) and antithrombotic (ATB) activities. The elongation of plasma clotting time in the APTT, thrombin time (TB) tests and the degree of inhibition of thrombin activity depend on the degree of sulfation (CC) of the 2,4-O-sulfated branches of fucose, which are part of fucosylated chondroitin E sulfates isolated from sea cucumbers Isostichopus badionotus and Pearsonothuria graeffei; whereas the inhibitory effect of fucans on the activation of factors X, XII and the appearance of a blood clot is attributed by the authors to the complete structure of polysaccharides.

In medical practice, there are several drugs based on heparin: native UFH, average molecular weight - MM ~ 15000), low molecular weight heparin (semi-synthetic NMH, MM ~ 6000) and ultra low molecular weight heparin (synthetic, MM <2000). The anticoagulant activity of heparins is due to the presence in the polysaccharide molecules of the antithrombin-binding pentasaccharide sequence {GlcNAc (6SO4) -GlcA-GlcNS (3SO4) -IdoA (2SO4) -GlcNS (6SO4)} [3]; this sequence activates antithrombin (a serine protease plasma inhibitor, serpin), after which the antithrombin inhibition rate of thrombin activity, factors IXa and Xa increases by 2-3 orders of magnitude. However, UFH to the same extent antithrombin-dependently inhibits the activity of thrombin and factor Xa (activity ratio aXa / aIIa = 0.9-1.2), LMWH to a greater extent the activity of factor Xa (aXa / aIIa = 1.5-2.5 ), and pentasaccharide - only the activity of factor Xa (no activity against thrombin). As a rule, with an increase in AK activity of heparins, their ATB activity increases. Pharmaceutical preparations of heparins are obtained from mammalian tissues (the intestinal mucosa of pigs or lungs of cattle).

However, since unfractionated heparin is also obtained from ruminant mammalian tissues, this anticoagulant may contain prions or chondroitin sulfate [4], and also provokes thrombocytopenia, bleeding, and osteoporosis.

The closest in essence and purpose to the present invention is an antithrombotic agent - cellulose sulfate, isolated from fir wood with a molecular weight of 3600, obtained by sulfation with chlorosulfonic acid in 1,4-dioxane according to the method described in patent No. 2404994 [5].

The disadvantages of this invention is the lack of antithrombotic activity. In addition, during the sulfation process, both the starting and sulfated polysaccharides are depolymerized, which can lead to a significant degradation of sulfated cellulose compared to the original cellulose and to a decrease in the antithrombotic and anticoagulant activity of sulfated cellulose.

It is known that with an increase in the molecular weight of polysaccharide sulfates from plant materials, anticoagulant activity sometimes increases.

The objective of the invention is to obtain compounds based on plant polysaccharide with greater antithrombotic activity.

The technical result of the invention is an effective tool for the prevention and treatment of thrombotic conditions based on chemically modified cellulose isolated from plant materials.

The technical result is achieved in that an antithrombotic agent based on sulfated cellulosic material is obtained, characterized in that it is cellulose sulfate obtained from partially hydrolyzed fir wood by sulfation with a pyridine complex with chlorosulfonic acid.

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, and which is the most common organic compound that is isolated from herbaceous and tree-like plants.

Obtaining an antithrombotic agent was carried out as follows.

The initial microcrystalline cellulose (MCC) was obtained according to the method described in the patent [6]. Sulfation of MCC fir was carried out by a preliminarily obtained complex of pyridine and chlorosulfonic acid (C ₅ H ₅ N · SO ₃ ) according to the procedure described in the patent [7]. The degree of substitution in the samples was found by the equation: C3s = 162 · ω _s / 3200-ω _s · 103, where ω _s is the sulfur content,% [8]. The sulfur content in MCC sulfate (SC) was determined by a modified method by burning in a stream of oxygen, followed by absorption of products of combustion with hydrogen peroxide and titration with alkali in the presence of a methyl red indicator [9]. The elemental composition of the MCC and SC was determined on a Flash EA-1112 elemental analyzer (Thermo Quest Italia). The average molecular weight of SC was determined by the viscometric method in accordance with the recommendations [10]. IR spectra were recorded on a Tensor-27 IR Fourier spectrometer (Bruker, Germany) in the wavelength range 400-4000 cm ^-1 . Spectral information was processed using the OPUS program (version 5.0). Solid samples for analysis were prepared as tablets in a KBr matrix (2 mg sample / 1000 mg KBr). The structural parameters of SCs were as follows: sulfation degree 1.65, sulfur amount 15.8%, dynamic viscosity 1.65 cPa, MM 80 kDa.

The specific anticoagulant (AK) activity of the obtained SC substance 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 (Renam, Moscow). Lyophilically dried human plasma was purchased at the Renam NGO. To calculate the antithrombin (aIIa) and anti-factor Xa (aXa) activities, the UFH calibration curves were used (Belmedpreparat OJSC; MM 15 kDa, aIIa activity 160 IU / mg, aXa activity 145 IU / mg).

The value of the antithrombin activity of SC (aIIa> 70 U / mg) in in vitro experiments allowed us to evaluate ex vivo ATB and hemorrhagic activities. These studies were carried out using laboratory animals (rats), which were obtained from the Stolbovaya RAMS nursery. All experiments were carried out in compliance with the "Rules for the use of 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 Russian Federation No. 708n of August 23, 2010. UFH (Belmedpreparat OJSC; MM 15 kDa, aIIa activity of 160 U / mg, aXa activity of 145 U / mg, aXa / aIIa = 0.91) was used as a comparison drug. To assess ATB activity, we used whether there was a model of venous thrombosis in rats based on an assessment of the formation of a thrombus induced by two factors - coagulation activation and stasis [11]. As a result, the venous thrombus consists of fibrin and erythrocytes. We have shown that with an increase in the dose of SC and UFH (Belmedpreparat OJSC) ; MM 15 kD, aIIa activity 160 IU / mg, aXa activity 145 IU / mg) increased ATB activity in simulated venous thrombosis in rats.To achieve 50% inhibition of thrombus growth, SC required 2 times more than UFH. Earlier, Wu B. and Xu J. showed that in order to reduce blood clots by 42.5%, when modeling rat arteriovenous shunt, administration of UFH at a dose of 0.6 mg / kg is necessary. Min S. et al. In a model of arterial thrombosis in mice, a 100% ATB effect was observed with the administration of UFH at a dose of 0.8 mg / kg [12].

Bleeding is the main complication of AK therapy, even when the therapeutic ranges of plasma anticoagulant activity are reached. Therefore, we evaluated the hemorrhagic activity of SC when administered intravenously to rats. The hemorrhagic activity of SC [13] by subcutaneous hemorrhage was determined when injected at the same doses as the comparison drug NFH (by aIIa activity). With an increase in the dose of SC and UFH (Belmedpreparat OJSC; MM 15 kDa, aIIa activity 160 U / mg, aXa activity 145 U / mg), hemorrhagic activity increased. However, in comparison with the control, depending on the dose, the concentration of hemoglobin in the blood hemolysates of rats after the administration of SC and NFH was 10–200 and 40–800 times higher, respectively. The 4-fold lower hemorrhagic effect of SC can be explained by 2 and 10 times lower than in heparin, inhibitory activities against thrombin and factor Xa, respectively.

The possibility of carrying out the claimed invention is shown by the following examples:

Example 1. Determination of the antithrombin activity of the substance SC: To a mixture of 0.06 ml of plasma and 0.06 ml of APTT reagent (NPO Renam), add 0.02 ml of a solution of SC or NFH (Belmedpreparat OJSC; aIIa = 160 PIECES / mg) at final concentrations of 0.08-1.0 μg / ml. After 3 minutes of incubation at 37 ° C, add 0.06 ml of a 0.025 M CaCl ₂ solution and on a Minilab 701-M coagulometer, fix the time (sec) for the appearance of a fibrin clot. In FIG. Figure 1 shows the effect of different concentrations of SC and NFH on the coagulation time of a human plasma in the APTT test (plasma coagulation time without anticoagulants 33.9 ± 1.1 sec). Comparing the readings of the SC concentration-time plasma clotting curves in 4 independent determinations with the UFH calibration curves (Belmedpreparat OJSC), we determine the antithrombin (aIIa) activity of the SC [14]; aIIa activity of SC was 83.4 ± 0.7 U / mg.

Example 2. Determination of the anti-factor Xa activity of the substance SC: To a mixture of 0.069 ml of plasma and 0.035 ml of a solution of factor Xa (test ReaKlot-Heparin, NPO Renam) add 0.01 ml of a solution of SC or NFH (Belmedpreparat OJSC; aXa = 145 IU / mg) at final concentrations of 0.07-2.36 μg / ml. After 2 min incubation at 37 ° C, add 0.06 ml of a 0.035 M CaCl ₂ solution and on the Minilab 701-M coagulometer we fix the time (sec) of the appearance of the fibrin clot. In FIG. Figure 2 shows the effect of SC and NFH on the time of coagulation of human plasma in the ReaKlot-Heparin test (time of coagulation of plasma without anticoagulants 30.6 ± 2.6 sec). Comparing the readings of the SC concentration-time plasma clotting curves in 4-independent determinations with the calibration curves of the NFH (Belmedpreparat OJSC), we determine the anti-factor Xa (aXa) of the SC activity [14]; aXa activity of the SC was 18.3 ± 0.2 U / mg.

Example 3. Antithrombotic activity (ATB) of SC is evaluated by modeling venous thrombosis according to Wessler S. et al. [11] in 41 rats of Wistar males weighing 250-350 g. For anesthesia, rats were administered intraperitoneally at a dose of 60 mg / kg. SC solutions in doses of 1, 3, 4 and 5 mg / kg are injected into the left jugular vein (the drug for comparison of UFH of Belmedpreparat OJSC is administered at doses of 1 and 2 mg / kg), and after 15 min - glass-activated human serum. Then, we thread a section of the opposite vein that was not used for AK insertion, and after 15 minutes we cut out the bandaged section of the vein and extract the contents into a small Petri dish. We evaluate the effectiveness of ATB activity: 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 is set according to a calibration curve constructed with a solution of fibrinogen (Behring, Germany) in concentrations from 10 to 400 μg / ml; 3) the size of the blood clot image in electronic JPG format in pixels. With an increase in the dose of SC, the following decreases: the form of blood clots from 3.33 to 0 points, the size of blood clots from 727 to 0 pc and the protein concentration in blood clot homogenates from 1.28 to 0 mg / ml (Table 1). With the introduction of SC at a dose of 5 mg / kg and UFH at a dose of 2 mg / kg, blood clots are not observed. We note significant differences in the form of blood clots between the indications with the introduction of saline and SC, starting with a dose of 3 mg / kg, and differences in blood clots and protein concentration in blood clot homogenates starting with a dose of 1 mg / kg.

Example 4. Hemorrhagic activity is determined on 68 rats of Wistar males weighing 200-250 g [13]. Anticoagulants (SC and NFG Belmedpreparat OJSC) in doses of 50, 100 and 200 aIIa IU / kg are injected into the jugular vein and after 30 s we cut the skin from the back. Under the skin to a depth of 10 cm, we introduce the sharp ends of the scissors, push the branches apart by 4 cm and in this position we remove the scissors, achieving separation of the skin from the underlying tissues. Place a 14 × 14 cm gauze napkin under the skin. After 30 minutes, remove the napkin and carry out hemolysis of red blood cells by adding 10 ml of distilled water. 2 hours after complete hemolysis of red blood cells, we measure the optical density of the samples on a spectrophotometer at a wavelength of 540 nm. The hemoglobin content in the samples is estimated by the calibration curve for rabbit hemoglobin (Sigma). By the concentration of hemoglobin in rat hemolysates, with an increase in dose from 50 to 200 aIIa IU / kg for SC (0.0022 ± 0.0011-0.0420 ± 0.0060 g / l) and UFH (0.0075 ± 0, 0026-0.1634 ± 0.0109 g / l) we observe significant differences (p = 0.0018-0.012; p = 0.0018, respectively) with the indications in the control (dose 0 mg / kg - 0.00020 ± 0, 00003 g / l) (Table 2). Significant differences in the indications between SC and UFH are observed when administered at doses of 100 and 200 aIIa IU / kg (p = 0.012).

The advantage of the claimed means, in comparison with the prototype is a large 1.5-fold antithrombotic activity, the value of which allows you to evaluate the antithrombotic effect on the model of experimental thrombosis.

Bibliography

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7. Patent RU No. 2426746, publ. 08/20/2011.

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Claims (1)

An antithrombotic agent based on sulfated cellulosic material, characterized in that it is cellulose sulfate with a molecular weight of 80,000 Da, obtained from partially hydrolyzed fir wood by sulfation with a complex of pyridine and chlorosulfonic acid.

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