RU2414907C2 - Pharmaceutical composition, possessing anticoagulant and cholesterol-lowering properties - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to medicine, in particular to pharmaceutical industry, namely to pharmaceutical composition, possessing anticoagulant and cholesterol-lowering properties, which contains heparin, immobilised on polymeric carrier by irradiation by gamma-rays or flux of accelerated electrons, and buffer mixture, with the following component ratio, wt %: heparin 4.0-5.0 polymeric carrier 8.0-10.0 buffer mixture - the remaining part. As polymeric carrier, applied is polyethylene oxide with molecular weight 400-4000 Da or dextran with molecular weight 20000-60000. As buffer mixture, used is 0.025 M sodium phosphate buffer with pH 7.5-8.2.

EFFECT: invention makes it possible to increase treatment efficiency due to increase of peroral bioavailability and extension of composition application range for treatment of diseases accompanied by phenomena of thrombus-formation due to cholesterol-lowering action in combination with anticoagulant properties.

5 cl, 4 ex

Description

The invention relates to medicine, in particular to the pharmaceutical industry, and can be used for the prevention and treatment of cardiovascular diseases, as well as for thrombosis and embolism of the main veins and arteries, blood vessels of the brain, eyes, heart and blood vessels.

Known release agent comprising chitosan immobilized on a polysaccharide selected from heparin, heparin sulfate and dextrin sulfate, capable of preventing or reducing unwanted adhesion of damaged tissue to adjacent or surrounding tissues during wound healing (Russian patent No. 2155592, publ. 09.10.2000).

However, the known release agent is a charged molecule and, due to the high hydrophilicity and negative charge of heparin and the positive charge of chitosan, is not able to penetrate the intestinal wall and is absorbed into the blood, which limits its bioavailability and can only be applied topically, therefore it does not have an anticoagulant prolonged action when administered subcutaneously or intramuscularly. In addition, in the release agent, a linear 1,4-linked polysaccharide consisting of V-D - glucosamine units, which has an immunostimulating effect that can cause adverse reactions, is used as a substrate with which heparin is covalently bound.

Closest to the proposed pharmaceutical composition are compositions comprising aliphilic derivatives of polysaccharides having anticoagulant properties, containing heparin immobilized with bile acids, sterols and alkanoic acids (US patent No. 6245753, CL A61K 31/727, publ. 2002).

The disadvantage of this technical solution is that the conjugate of heparin with bile acids, sterols and alkanoic acids has a low prolonged anticoagulant effect, because Bile acids are products of animal origin, so there is a rapid enzymatic destruction of the conjugate and its relatively rapid elimination from the body (after 4 hours, the anticoagulant activity of the blood begins to decrease), which reduces the bioavailability of the conjugate and, consequently, the effectiveness of the treatment.

Disclosure of invention

The present invention is the creation of a new pharmaceutical composition, which allows to increase the effectiveness of treatment by increasing the oral bioavailability and expanding the scope of the composition for the treatment of diseases accompanied by thrombosis due to hypocholesterolemic action in combination with anticoagulant properties.

The problem is achieved by the proposed pharmaceutical composition having anticoagulant and hypocholesterolemic properties, including the following components, wt.%:

heparin 4-5 polymer carrier (polyethylene oxide with a molecular weight of 400-4000 Yes or molecular dextran weighing 20000-60000 Yes) 8-10 buffer mixture rest

As a buffer mixture using 0.025 M sodium phosphate buffer with a pH of 7.5-8.2.

The proposed composition is subjected to irradiation, mainly gamma radiation or a stream of accelerated electrons with an energy of 2 MeV at a dose of 0.5-1.5 Mrad to immobilize heparin on polyethylene oxide or dextran.

The heparin introduced into the pharmaceutical composition when exposed to ionizing radiation binds to a polymer carrier - polyethylene oxide (MM 400-4000 Da) or dextran (MM 20000-60000 Da), forming an immobilized form that is resistant to enzymatic destruction in the body and is slowly excreted from the body, due to which increases the prolonged effect of the drug and its oral bioavailability, which increases the effectiveness of the treatment.

Heparin immobilized on a polymer carrier has hypocholesterolemic properties, because binds to blood lipoproteins enriched in cholesterol, and is excreted from them from the blood, which allows to expand the scope of the composition for the treatment of diseases accompanied by thrombosis.

The buffer mixture in the proposed composition creates the conditions for reducing the charge of immobilized heparin and, therefore, increases its bioavailability when absorbed in the gastrointestinal tract.

It has been experimentally established that only with such a ratio of components in the inventive tool, the necessary technical result is achieved.

When the concentration of heparin is less than 4.0 wt.%, The activity of the proposed product is insufficient for the effective action of the claimed properties, and when the concentration of heparin is more than 5.0 wt.%, The risk factor for reducing coagulation of blood-blocking thrombin biosynthesis and reducing platelet aggregation increases.

As the polymeric carrier for immobilizing heparin, it is preferable to use polyethylene oxide with MM 400-4000 Da or dextran with MM 20000-60000 Da. When the concentration of polyethylene oxide is less than 8.0 wt.%, The pharmacological effectiveness of the agent decreases, and when the concentration of polyethylene oxide is more than 10.0 wt.%, The conjugate precipitates.

The implementation of the invention

The proposed pharmaceutical composition is prepared as follows.

Example 1

The reaction mixture is prepared by dissolving heparin and polyethylene oxide with a molecular weight of 400-4000 Da in 0.025 M sodium phosphate buffer solution pH 7.6-8.2 (wt.% Heparin and polyethylene oxide of the total composition are 4% and 8%, respectively) . The resulting mixture is subjected to irradiation with gamma rays or a stream of accelerated electrons (with an energy of 2.0 MeV) at a dose of 0.5-1.5 Mrad. After irradiation, the solution is packaged in 10 ml in bottles with a capacity of 15 ml.

Get heparin immobilized on polyethylene oxide. The activity of immobilized heparin in the resulting composition in one bottle is 25,000 IU (2500 IU / ml). The composition is a colorless homogeneous solution.

Example 2

The reaction mixture is prepared by dissolving heparin and dextran with a molecular weight of 20000-60000 Yes in a 0.025 M sodium phosphate buffer solution pH 7.6-8.2 (wt.% Heparin and dextran of the total weight of the composition are 5% and 10%, respectively) . The resulting mixture is subjected to irradiation with gamma rays or a stream of accelerated electrons (with an energy of 2.0 MeV) at a dose of 0.5-1.5 Mrad. After irradiation, the solution is packaged in 10 ml in bottles with a capacity of 15 ml.

The result is heparin immobilized on dextran. The activity of immobilized heparin in one bottle is 25,000 ME (2500 IU / ml). The composition is a colorless homogeneous solution.

Similarly obtained compositions containing wt.% Heparin and polyethylene oxide of the total weight of the composition, equal to 5% and 10%, respectively, and also containing wt.% Heparin and dextran of the total weight of the composition, equal to 4% and 8%, respectively).

Example 3

In order to experimentally verify the hypocholesterolemic effect during intragastric administration of the heparin drug immobilized on polyethylene oxide, we used a model of experimental hyperlipidemia in mice of the inbred C57 / B1 line arising from the intraperitoneal administration of poloxamer-407. The introduction of poloxamer-407 is a modern model of hyperlipimia used to test lipid-lowering drugs. A solution of poloxamer-407 (Fluka) was administered to mice weighing 20 g intraperitoneally at a dose of 15 mg / 20 g of mass in saline in the evening (at 18 h). After 15 hours (in the morning at 10 hours), a heparin drug immobilized with polyethylene oxide at a dose of 50 and 100 IU of heparin per 20 g of mouse was injected intragastrically. As an additional control, free heparin was introduced in a similar manner in similar concentrations. After 8 hours, the animals were killed by decapitation and blood serum was obtained by centrifugation at 3000 rpm for 10 minutes. In the blood serum, the content of total cholesterol was determined by enzymatic method using the Biocon kit (Germany).

The research results are presented in table 1. As can be seen from the table, the administration of poloxamer-407 led to an increase in cholesterol by 5.86 times compared with the level of intact animals (administration of saline). Intragastric administration of pure heparin to hypercholesterolemic animals did not have a significant effect on serum cholesterol (experimental groups 3 and 4). The data obtained with high reliability indicate that intragastric administration of the drug heparin immobilized with polyethylene oxide with MM 400-4000 Yes, unlike pure heparin, the dose - dependently lowers blood cholesterol. At doses of 50 and 100 IU / 20 g of animal weight, the concentration of cholesterol in the blood of hyperlipidemic mice is reduced by 3.1 and 5.35 times, respectively. Moreover, at a dose of 100 IU, the concentration of cholesterol returns to the control level. The reliability of the observed phenomenon is confirmed by three independent experiments.

Table 1 Group number The number of animals in the group Experiment Conditions The administered dose in units / 20 g of mouse weight The concentration of cholesterol in serum in mmol / l (M ± m) one 8 Intact animals 3.64 ± 0.32 2 10 The introduction of poloxamer-407 15 mg 21.33 ± 2.43 3 10 Introduction of non-immobilized heparin 50 units 22.74 ± 3.86 four 10 Introduction of non-immobilized heparin 100 units 20.85 ± 2.75 5 10 The introduction of immobilized heparin 50 units 6.87 ± 0.89 * 6 10 The introduction of immobilized heparin 100 units 3.98 ± 0.58 ** 7 10 The introduction of heparin associated with a hydrophobic agent (prototype) 50 units 20.32 ± 4.12 8 10 The introduction of heparin associated with a hydrophobic agent (prototype) 100 units 18.95 ± 4.75 Note: * - reliable values in comparison with group 2 (p <0.02); ** - reliable values in comparison with group 2 (p <0.001).

Example 4

The anticoagulant properties of heparin immobilized on polyethylene oxide (MM 400-4000 Da) were studied in mice of the C57B1 line inbred mouse. Heparin was introduced intragastrically, immobilized on polyethylene oxide with MM 400-4000 Da, at a dose of 100 IU / 20 g of mouse. After 2, 4, 8, 10, 14, and 24 hours after administration, the animals were decapitated and the blood coagulation rate was determined using an ATVT kit. The research results are presented in table 2.

table 2 Experimental groups Blood coagulation rate, in s, at various intervals after administration in hours 0 2 four 8 10 fourteen 24 Heparin associated with a hydrophobic agent (prototype) 23 ± 2.5 68 ± 13.4 168 ± 2.5 54 ± 5.8 29 ± 3.2 28 ± 2,3 21 ± 3,5 Heparin immobilized on polyethylene oxide (invention) 22 ± 2.1 76 ± 9.8 134 ± 14.1 126 ± 11.4 96 ± 10.1 69 ± 8.2 64 ± 7.2 Protosubtilin immobilized on polyethylene oxide and dextran 23 ± 2.6 26 ± 2.4 24 ± 3.2 22 ± 2.4 21 ± 2.5 22 ± 2.2 23 ± 2.1

The introduction of heparin immobilized on polyethylene oxide with MM 400-4000 Yes, led to a significant increase in the rate of blood coagulation in all studied time intervals compared with the prototype. The maximum value of the blood coagulation rate after administration of heparin immobilized on polyethylene oxide with MM 400-4000 Yes, in accordance with the present invention and immobilized heparin, disclosed in the closest analogue, reached after 4 hours. However, after 8 hours, the blood coagulation rate in the prototype decreased by 3 times compared with the maximum value. After 10 hours of administration, the blood coagulation rate in the experiment by the closest analogue fell to the initial value (23 ± 2.5 s). 10 hours after the administration of heparin immobilized on polyethylene oxide with MM 400-4000 Da, in accordance with the present invention, the blood coagulation rate remained high, exceeding the initial values by 4 times. After 14 and 24 hours, the rate of blood coagulation decreased slowly, exceeding the initial indications by 3 times.

Similar studies were conducted with the agents of the present invention, which are heparin immobilized on dextran (MM 20000-60000 Da).

Studies have shown that in contrast to the immobilized form of heparin according to the prototype, heparin immobilized on polyethylene oxide (MM 400-4000 Da) or dextran (MM 20000-60000 Da), in accordance with the present invention has a higher prolonged anticoagulant effect when administered orally.

Thus, the proposed pharmaceutical composition due to the presence in its composition of heparin immobilized on polyethylene oxide (MM 400-4000 Da) or dextran (MM 20000-60000 Yes), allows to increase the effectiveness of treatment and expand the scope for the treatment of diseases accompanied by thrombosis, for due to hypocholesterolemic action in combination with anticoagulant properties.

Claims (5)

1. A pharmaceutical composition having anticoagulant and hypocholesterolemic properties, containing heparin immobilized on a polymer carrier by irradiation with gamma rays or a stream of accelerated electrons, and a buffer mixture in the following ratio of components, wt.%:
Heparin 4.0-5.0 Polymer carrier 8.0-10.0 Buffer mixture rest 2. The pharmaceutical composition according to claim 1, characterized in that polyethylene oxide with a molecular weight of 400-4000 Da is used as a polymeric carrier. 3. The pharmaceutical composition according to claim 1, characterized in that the dextran with a molecular weight of 20,000-60000 Da is used as a polymeric carrier. 4. The pharmaceutical composition according to claim 1, characterized in that 0.025 M sodium phosphate buffer with a pH of 7.5-8.2 is used as a buffer mixture. 5. The pharmaceutical composition according to claim 1, characterized in that the immobilization of heparin is carried out by a stream of accelerated electrons or gamma radiation in doses of 0.5-1.5 Mrad.