RU2454237C1 - High-molecular heparin composition with anticoagulant, anti-platelet, fibrinolytic and fibrin depolymerisation properties - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmacology, and represents a composition of high-molecular heparin and fish oil Eiconol exhibiting properties for oral application differing by the fact that it additionally contains leucine in the following amounts of the ingredients at 90-120 IU of heparin activity: leucine - 0.56-0.58 mg, Eiconol -113-115 mg.

EFFECT: invention provides more evident anticoagulant, anti-plate, fibrinolytic and fibrin depolymerisation action in single oral administration.

3 cl, 4 ex, 1 tbl

Description

The invention relates to medicine and pharmacology and for the creation of agents based on high molecular weight heparin with anticoagulant, antiplatelet and fibrinolytic effects on the body.

Most often in medicine, drugs based on heparin and its derivatives are used as anticoagulant agents.

High molecular weight heparin (VMH) is a natural anticoagulant and refers to direct-acting anticoagulants, i.e. directly affects blood coagulation factors. Heparin consists of residues of glucuronic acid and glucosamine esterified with sulfuric acid, not only has an anticoagulant effect, but to some extent it can activate the fibrinolytic properties of blood and improve coronary blood flow. The introduction of heparin into the body is accompanied by a certain decrease in the content of cholesterol and β-lipoproteins in the blood serum

The anticoagulant effect of heparin is manifested in vitro and in vivo when administered intravenously, intramuscularly and subcutaneously. Heparin acts quickly, but relatively briefly.

High molecular weight heparin is used in the prevention and treatment of various thromboembolic diseases and their complications, for example, to prevent or limit thrombosis in acute myocardial infarction, thrombosis and embolism of the main veins and arteries, vessels of the brain and eyes.

However, at the same time, a number of negative properties are inherent in heparin, such as thrombocytopenia and immediate allergic reactions (urticaria, angioedema or bronchospasm), and in case of an overdose - the possibility of hemorrhage.

[Clinical Biochemistry / Ed. V.A. Tkachuk. 2nd ed., Rev. and add. M.: GEOTAR-MED, 2004. 512 p.].

Known anticoagulant-fibrinolytic agent, including the peptide "Semax" and heparin. The name of the peptide "Semax" approved by the decision of the Pharmacological Committee of the Ministry of Health of the Russian Federation from 11.11.93 [US Pat. RF №2138289, А61К 31/725, publ. 1999.09.27].

Known thrombolytic complex of heparin with the enzyme trypsin obtained at a weight ratio of 1: 6. However, the use of trypsin gives this product toxic proteolytic properties. Moreover, the resulting drug is only active when given intravenously or intramuscularly.

[Mansfeld V, Hladovec J. Studien über Reactionen des Kristallisierten Trypsins. II. Beziehung zwischen der Aktivität des Heparins und Trypsins - "Collection of Czechoslovak chemical communication", 1957, v. 21, No. 5, p. 1209.]

These funds have a different mechanism of anticoagulant action than the mechanism of action of heparin, however, the method of obtaining them is complex, and, in addition, antiplatelet and anticoagulant action is not enough.

High molecular weight heparin is often used in combination with enzymatic fibrinolytic drugs.

Known anticoagulant-fibrinolytic agent of the complex of high molecular weight heparin with the proteolytic enzyme thrombin. The drug is effective for intravenous and intramuscular injections. However, the short duration of the drug’s action is 1.5-2.0 hours and the presence of side effects does not allow its widespread use in medical practice. [Journal. "Biochemistry of animals and humans", "Non-enzymatic fibrinolysis", Lyapina L.A. and others. Kiev, 1982, No. 6, pp. 64-73].

A known therapeutic composition having anticoagulant activity based on complexes of heparin with substances that enhance the anticoagulant effect, in particular with amino acids.

The specified composition is obtained as follows. An aqueous solution of sodium heparinate is successively applied to columns with a cation exchange and anion exchange resin, after which an aqueous solution of the amino acid in the ratio of 1: 1.1 is added to the resulting filtrate with stirring, the resulting complex is precipitated with an organic solvent - alcohol or acetone, and the precipitate is separated by centrifugation, after which freeze-dried.

The yield based on the initial anticoagulant activity is from 100 to 120%. Thus obtained agent exhibits anticoagulant and minor antilipidemic properties.

The disadvantages of the known composition include the complexity of obtaining the composition, the lack of fibrinolytic and antiplatelet properties of the resulting products. Moreover, the resulting preparations are unstable and after 17-20 days of storage, a loss of activity occurs when stored at room temperature.

[US Patent No. 3577534, Cl. C08B 37/10, publ. 1971.05.04].

The closest composition to its pharmacological action is an antithrombotic composition based on high molecular weight heparin, including fish oil Eikonol.

[US Pat. RF №2359681, А61К 31/727, А61К 35/60, А61Р 7/02, publ. 2009.06.27].

This patent uses syntileit domestic heparin and Trinita fish oil Eikonol in the form of its 25-45% solution in edible vegetable oil in the amount of 1 ml per 80-120 ME of antithrombin activity of high molecular weight heparin. Both liquid and dry high molecular weight heparin are used.

The specified tool is obtained by mixing Eikonol and liquid VMG in a volume ratio of Eikonol: VMG equal to 1: 0.018-0.022, or dry VMG and Eikonol in quantities of 0.8-1.2 parts by weight. heparin (VMG) per 1 vol. Eikonola.

To obtain the composition, the original commercial preparation of Eikonol (E) is diluted with refined corn oil to a concentration of 25-45% and mixed with 80-120 ME VMG, and is stirred periodically every 30 minutes for 1-2 hours at room temperature.

The drug has anticoagulant, antiplatelet, fibrinolytic, fibrindepolymerization effects with its repeated oral administration to rats.

The disadvantage of this tool is its low anticoagulant (127.5%), fibrinolytic (140%) and fibrindepolymerization (133%) effects; in connection with which it is necessary its multiple (at least 4 times) introduction to obtain a wide range of anticoagulant effects; dilution of commercial eikonol with edible oil is necessary.

All of the above allows us to conclude that the search for new effective and safe anticoagulants and fibrinolytics remains extremely relevant to date.

The present invention is based on the task of creating a more effective composition with a more pronounced anticoagulant, fibrinolytic, fibrin-depolymerization and antiplatelet effect with its single oral administration.

This object is achieved by creating a composition for oral administration with anticoagulant, antiplatelet, fibrinolytic and fibrin-depolymerization properties based on high molecular weight heparin, fish oil Eikonol and leucine with the following amounts of components based on 90-120 IU of heparin activity:

leucine - 0.56-0.58 mg eikonol - 113-115 mg

The composition when using dry high molecular weight heparin contains heparin: leucine: Eikonol in the ratio (parts by weight):

heparin - 0.9-1.1 leucine - 0.73-0.76 eikonol - 147-150

The composition when using liquid high molecular weight heparin contains leucine and Eikonol in the following amounts per 1 ml of liquid high molecular weight heparin:

leucine - 28.4-29 mg eikonol - 5680-5700 mg

The composition uses Eikonol fish oil in the form of its commercial preparation.

Leucine is one of the essential amino acids that are not synthesized by animal and human cells and enter the body as part of food proteins. Leucine is widespread in animals and plants; in humans, found in the pancreas, spleen, in the liver, kidneys, as part of serum proteins, in muscle tissue. The daily human need for leucine is 4-6 g. Leucine is involved in ensuring nitrogen balance, in the metabolism of proteins and carbohydrates, the construction and development of muscle tissue, strengthening the immune system, etc.

Eikonol, produced according to TU 9197-00429388404-08 from pelagic fish tissues, is a product rich in essential omega-3 polyunsaturated acids with up to 30% content. Eikonol is recommended for the treatment of allergic lesions of the skin and the gastrointestinal tract and lowering the level of total cholesterol and triglycerides in the blood.

To obtain the composition, 88,000 IU, approximately (700 mg) of dry, high molecular weight heparin (G) and a commercial preparation of the amino acid leucine (L) in an amount of 500 mg are added to the initial commercial preparation of Eikonol (E) in an amount of 100 g, which corresponds to a molar ratio of G: L, equal to 1: 3, and mix them for 35-60 minutes at room temperature. The molar ratio of 1: 3 is optimal for the manifestation of the maximum anticoagulant activity of the complex of heparin with L-leucine, which is proved by computer simulation according to the universal computer program for calculating chemical equilibria AUTOEQUIL, pH meter data of physiological solutions: heparin - L-leucine and calcium - heparin- L-Leucine.

The resulting composition was investigated as follows.

Under in vitro conditions, the starting composition is checked for fibrindepolymerization activity (non-enzymatic fibrinolytic activity) using fibrin films unstabilized by factor XIIIa. When the composition is added to normal rat blood plasma, the anticoagulant and fibrinolytic (enzymatic and non-enzymatic nature) properties in the blood plasma are checked.

In vivo, 10 male white laboratory rats with a body weight of 200-220 were used. The first group of rats received an oral composition (experiment E + G + L) 0.05 ml each, and the second received a similar saline solution (control). Blood was taken for analysis 1 hour after a single oral administration of drugs.

The following were determined in blood samples: anticoagulant activity according to the activated partial thromboplastin time test (APTT), total (SFA) and non-enzymatic (NF), enzymatic (FF) fibrinolytic activity and platelet aggregation.

The following materials illustrate the invention.

Example 1

To 1 g of the original commercial preparation of Eikonol (E) add 880 ME dry high molecular weight heparin (G) (7 mg) and commercial preparation of the amino acid leucine (L) in an amount of 5 mg, the ratio (weight.) G: L: E = 1: 0 , 71: 149 or per 100 IU of heparin 114 mg (parts by weight) of eikonol and 0.57 mg (parts by weight) of leucine, stirring them for 35 minutes at room temperature, then the mixture is left in the refrigerator at 4-6 ° C before use.

In vitro, the resulting composition has a fibrindepolymerization effect of 34 mm ² (in the control, 0 mm ² ), and when added to the blood plasma of rats, it exhibits a total fibrinolytic activity of 136%, fibrindepolymerization activity of 120%, anticoagulant activity of 227.7 % of control taken as 100%.

The resulting composition, when administered orally to rats weighing 200 g at a dose of 0.05 g (45 IU heparin) in the blood plasma, exhibits 190% fibrindepolymerization activity, 189% of the total fibrinolytic activity, 138.8% anticoagulant activity, and a 38% decrease in platelet aggregation. Oral E alone has moderate total (112%) and non-enzymatic (116%) fibrinolytic activity. Under these experimental conditions, Eikonol does not exhibit anticoagulant and antiplatelet action against platelets (i.e., antiplatelet). The composition of Eikonol with heparin in a single oral administration to rats weighing 200 g at a dose of 0.05 g (45 IU heparin) in the blood plasma reveals 121% of fibrindepolymerization activity, 123% of the total fibrinolytic activity, 112% of anticoagulant activity and a decrease in platelet aggregation by 12% compared to control (administration of saline to rats).

So, the resulting composition Eikonol + heparin + leucine has a pronounced total and non-enzymatic fibrinolytic effect, as well as anticoagulant and antiplatelet action in the bloodstream after its single oral administration to animals (table).

Example 2

To 1000 mg of the original commercial preparation of Eikonol (E) add 125 IU of dry high molecular weight heparin (G) (1 mg) and a commercial preparation of the amino acid leucine (L) in an amount of 5 mg (per 100 IU of heparin activity, 4 mg is required (weight.h) .) leucine and 800 mg (parts by weight) of eikonol or (ratio (weight) G: L: E = 1: 5: 1000), stirring them for 45 minutes at room temperature, then the mixture is left in the refrigerator at 4 -6 ° C before use.

The resulting composition (the dose of heparin is 7 times less than in example No. 1) with a single oral administration to rats weighing 200 g at a dose of 0.05 g (5 IU of heparin) detects 118% of fibrindepolymerization activity in blood plasma, 120% of the total fibrinolytic activity and 108% anticoagulant activity and does not detect antiplatelet activity. One E at single oral administration had a low total (112%) and non-enzymatic (116%) fibrinolytic activity. Under these experimental conditions, E did not exhibit anticoagulant and antiplatelet action.

The composition of Eikonol + heparin at a dose of 7 times less than in example No. 1, with a single oral administration to rats weighing 200 g at a dose of 0.05 g (5 IU of heparin) in the blood plasma reveals 116% fibrindepolymerization activity, 114% of the total fibrinolytic activity and not detects anticoagulant and antiplatelet activity.

The composition Eikonol + heparin + leucine obtained in the ratios indicated in Example 2 has a weak total (120%) and non-enzymatic fibrinolytic (118%) effect and does not have anticoagulant and antiplatelet effect in the bloodstream after its single oral administration to animals.

Example 3

To 1 g of the original commercial preparation of Eikonol add 5000 ME (approximately 40 mg) of high molecular weight dry heparin and commercial preparation of the amino acid leucine (L) in an amount of 5 mg (per 100 ME of heparin activity, 0.1 mg (parts by weight) of leucine is needed and 20 mg (parts by weight) of eikonol or (ratio (weight.) G: L: E = 1: 0.125: 25), stirring them for 60 minutes at room temperature, then the mixture is left in the refrigerator at 4-6 ° C before use.

The resulting composition, upon single oral administration to rats weighing 200 g at a dose of 0.05 g (250 IU of heparin), reveals 150% anticoagulant activity in the blood plasma, but does not show fibrindepolymerization activity, total fibrinolytic and antiplatelet activity. Oral E alone had moderate total (112%) and non-enzymatic (116%) fibrinolytic activity. Under these experimental conditions, E did not exhibit anticoagulant and antiplatelet action against platelets (antiplatelet). A single oral administration of the composition of Eikonol + heparin at a dose of 250 ME only contributed to the manifestation of anticoagulant plasma activity of 150%, but neither fibrin-polymerization, nor total fibrinolytic nor antiplatelet activity was established.

The resulting composition (the dose of heparin is 5 times higher than in example No. 1) has a high anticoagulant effect, but does not have a total and non-enzymatic fibrinolytic and antiplatelet effect in the bloodstream after its oral administration to animals.

Example No. 4

To 1 g of the original commercial preparation of Eikonol (E) add 880 IU of liquid high molecular weight heparin (G) (0.176 ml) and a commercial preparation of the amino acid leucine (L) in an amount of 5 mg, (ratio G: L: E = 1 (vol.h. ): 28.4 mg (parts by weight): 5682 mg (parts by weight) or per 100 IU of heparin 0.57 mg (parts by weight) of leucine and 114 mg (parts by weight) of eikonol, mixing them for 35 min at room temperature, then the mixture is left in the refrigerator at 4-6 ° C until use.

In vitro, the resulting composition has a fibrindepolymerization effect of 35 mm ² (in the control, 0 mm ² ), and when added to the blood plasma of rats, it exhibits a total fibrinolytic activity of 138%, fibrindepolymerization activity of 125%, anticoagulant activity of 221 % of control taken as 100%.

The resulting composition, when administered orally to rats weighing 200 g at a dose of 0.05 g (45 IU heparin), exhibits 190% fibrin-depolymerization activity, 185% of total fibrinolytic activity, 140% anticoagulant activity, and a 40% decrease in platelet aggregation in blood plasma. Oral E alone has moderate total (112%) and non-enzymatic (116%) fibrinolytic activity. Under these experimental conditions, Eikonol does not exhibit anticoagulant and antiplatelet action against platelets (i.e., antiplatelet). The composition of Eikonol with heparin in a single oral administration to rats weighing 200 g at a dose of 0.05 g (45 IU heparin) in blood plasma reveals 121% fibrindepolymerization activity, 123% of the total fibrinolytic activity, 112% anticoagulant activity and a 12% decrease in platelet aggregation compared to control (administration of saline to rats).

So, the resulting composition Eikonol + heparin + leucine has a pronounced total and non-enzymatic fibrinolytic effect, as well as anticoagulant and antiplatelet effect in the bloodstream after its single oral administration to animals.

Indicators of plasma and platelet hemostasis after oral administration to animals of one eikonol (E), a mixture of eikonol with heparin (E + G) and the composition of eikonol with heparin and leucine (E + G + L) (M ± m) are shown in the table.

Statistical indicators are calculated relative to the corresponding control samples (* p <0.05, ** p <0.01).

Thus, the Eikonol + heparin + leucine composition has in vitro and in vivo anticoagulant (at least 138%), fibrinolytic (at least 185%), fibrin-depolymerization (at least 190%) and antiplatelet effect (decrease in platelet aggregation by not less than 35 %) with its single oral administration.

In the body, the target product is able to prevent and reduce the level of thrombosis during oral administration. With a single oral administration of E + G + L, an increase in the anticoagulant, fibrinolytic activity of plasma and a decrease in platelet aggregation in the blood of animals were found. The composition can be used to prevent and correct thromboembolic disorders and to limit thrombosis in the main vessels (arteries and veins).

The composition is stable during storage at 4-6 ° C, non-toxic with the oral route of administration into the body, the duration of action of the drug in the body is up to 6-8 hours. The method of obtaining it is economically advantageous.

Table Experience Conditions APTT,% SFA,% NF,% Platelet Aggregation,% frequency of introduction Introduction E 99 ± 3.8 112 ± 8.6 116 ± 2.7 115 ± 3.3 one Introduction E + G 112 ± 4.3 123 ± 1.5 121 ± 1.8 88 ± 7.9 one Introduction E + G prototype 127.5 ± 8.0 140 ± 11.2 133 ± 8.7 76 ± 5.9 four Introduction E + G + L example number 1 138.8 ± 7.8 189 ± 10.9 190 ± 14.3 62.2 ± 9.2 one example number 2 108 ± 3.3 120 ± 1.0 118 ± 1.8 100 ± 6.9 one example number 3 150 ± 5.6 100 ± 2.8 108 ± 7.8 99 ± 6.5 one example number 4 140 ± 6.6 185 ± 8.9 190 ± 10.3 60.5 ± 6.0 one Control: the introduction of saline 100 ± 9.2 100 ± 1.6 100 ± 9.4 100 ± 10.5 one

Claims (3)

1. A composition based on high molecular weight heparin and Eikonol fish oil, which has anticoagulant, antiplatelet, fibrinolytic and fibrindepolymerization properties for oral use, characterized in that it additionally contains leucine with the following amounts of components per 90-120 IU of heparin activity, mg:
leucine 0.56-0.58 eikonol 113-115 2. The composition according to claim 1, characterized in that they use dry high molecular weight heparin, leucine and Eikonol in the ratio, parts by weight:
heparin 0.9-1.1 leucine 0.73-0.76 eikonol 147-150 3. The composition according to claim 1, characterized in that use liquid high molecular weight heparin with the following amounts of components, mg per 1 ml of liquid high molecular weight heparin:
leucine 28.4-29 eikonol 5680-5700