RU2522879C1 - Biodegradable haemostatic therapeutic agent for control of capillary and parenchymatous bleedings - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a biodegradable haemostatic therapeutic agent for bleeding. Using the prepared haemostatic agent according to the declared method provides a rate of control of bleeding making 45±2 seconds.

EFFECT: higher rate of control of bleeding.

4 cl, 2 tbl

Description

The invention relates to pharmacy, and specifically to methods for producing hemostatic (hemostatic) preparations based on partially oxidized cellulose.

The drugs mentioned above are known. Thus, in EPO application No. 1153620, a hemostatic dressing made of oxidized cellulose SURGICEL (Johnson and Johnson company) containing 1 to 10,000 ppm of iron ions (3+) is described, and soluble hemostatic is known from EPO application No. 0468114 material in the form of a cellulose web oxidized with monochloracetic acid and sodium hypochlorite to introduce COOH carboxyl groups.

EPO application No. 0659440 describes a method for producing hemostatic based on oxidized cellulose containing 0.5-4.0% calcium, as well as thrombin, fibrinogen and / or antifibrinolytic. The disadvantages of this method include the use of cellulose, the mild oxidation state of which, with the conversion of CH ₂ OH groups to carboxyl (COOH), does not allow to obtain a material that provides the possibility of strong binding of ingredients to it: only Ca ^{2+ ions} are associated with COOH groups ionic forces, while the remaining components are in the form of a physical (mechanical) mixture. In addition, such materials are only difficult to grind, for example, into powder.

A known method of producing a substance with a hemostatic effect, which includes mixing in an aqueous medium a carrier - partially oxidized cellulose to dialdehyde cellulose (DAC) - with coagulation factors, for example, thrombin and fibrinogen, gelatin, ε-aminocaproic acid and lysozyme are additionally added to these substances and as partially oxidized cellulose, dialdehyde cellulose in the form of a web having an oxidation state, i.e. the content of aldehyde groups, from 4 to 6%, with the following ratios between the components:

 dialdehyde cellulose 1 g  fibrinogen 18/22 mg  gelatin 27/33 mg  ε-aminocaproic acid 45/55 mg  lysozyme 9.5 / 10.5 mg  thrombin 350 units  water 6.5 ml

The components are maintained until the drugs are fully covalently bound to dialdehyde cellulose, followed by drying and grinding to powder. In order to prevent the interaction of thrombin and fibrinogen, the process of obtaining the drug is carried out in two stages. Separately, a solution of fibrinogen, ε-aminocaproic acid and half of the total amount of gelatin in half of the total amount of water and a solution of thrombin, lysozyme and the remaining amount of gelatin in the remaining amount of water are prepared separately; the solutions are kept for 3-4 hours in half the amount of dialdehyde cellulose , intermediates are squeezed, dried in air and subjected to joint grinding. See, for example, RF patent No. 2235539, 2003 (analogue).

The specified technical solution is not without drawbacks. First, blood products - thrombin and fibrinogen - are very expensive and scarce. In addition, more and more patients in the world refuse to use drugs obtained from organs and tissues of animals or donated blood. Secondly, in order to avoid interaction between thrombin and fibrinogen in an aqueous solution, their immobilization is carried out separately, then the canvases are dried and co-milled to a powder. This complicates and increases the cost of the process. The time to stop bleeding with this drug in the experiment was 110 seconds.

Known textile material to stop bleeding and a method for its preparation of the following composition:

DAC with an oxidation state of 6.5 ± 0.33 - 1 g

gelatinol - 60 ± 3 mg (0.75 ml of 8% solution)

ε-aminocaproic acid - 5 ± 0.25 mg

lysozyme - 5 ± 0.25 mg

water - 5.75 ml

The time to stop bleeding when tested on a group of volunteers of 25 people with punctured, cut wounds, bullet and mine-fragmentation wounds and other injuries accompanied by local bleeding was 60 ± 3 seconds (see, for example, RF patent No. 2380117, 2007 ( prototype).

The Israeli analogue differs from the previous one in that it uses either ε-aminocaproic acid or tranexamic acid, which are structural analogues, in equal quantities as a hemostatic ingredient. The time to stop bleeding with materials is the same and does not differ from the Russian prototype (see, for example, State of Israel Patent Office, Sertificate of Patent No. 178867, 2006 (analogue).

The known solution is not without flaws. An increase in the oxidation state of DAC to 6.5% did not lead to a change in the rate of stopping bleeding. On this basis, the authors concluded that 6.5% oxidation is sufficient to link the aldehyde groups of the components (and all components of the last two formulations have free amino groups) with DAC by covalent chemical azomethine bonds. Therefore, to oxidize cellulose to DAC with a degree above 6.5% is irrational. However, further in-depth studies have shown the failure of such a conclusion. Samples of hemostatic materials were prepared at the DAC with oxidation states of 12, 15 and 21%, containing hemostatic components in the following proportions:

DAC with oxidation states of 12, 15 and 21% - 1 g

gelatinol - 60 ± 3 mg

lysozyme - 5 ± 0.25 mg

epsilon aminocaproic acid - 50 ± 0.25 mg

tranexamic acid - 5 ± 0.25 mg

The time to stop bleeding in all cases was 45 ± 2 seconds. Comparative characteristics of hemostatic materials and their relationship with the degree of oxidation of the DAC and composition are shown in table 1.

Table 1 Hemostatic properties of materials The degree of oxidation,% Coagulation factors, mg per 1 g of DAC Hydrolytic degradation rate constant Time to stop bleeding, sec Full resorption time, days According to the patent of the Russian Federation No. 2235539 4-6% fibrinogen 18-22 18 × 10 ^-5 min ^-1 110 twenty gelatin 27-23 aminocaproic acid 45-55 lysozyme 9.5-10.5 thrombin 350 units. According to the patent of the Russian Federation No. 23 80,117 6.5 gelatin 60 21 × 10 ^-5 min ^-1 60 ± 3 fifteen aminocaproic acid 50 lysozyme 5 Israeli patent the same or with a replacement 21 × 10 ^-5 min ^-1 60 ± 3 fifteen

No. 178867 6.5 aminocaproic acid per transcamic acid in the same amount According to the proposed decision 12 aminocaproic acid is administered in an amount of 50 mg / g, lysozyme - 5 mg / g 33 × 10 ^-5 min ^-1 45 ± 2 10 eighteen the same 39 × 10 ^-5 min ^-1 45 ± 2 8

On this basis, the final conclusion was made that the oxidation state of DAC of 12% is sufficient to bind all amine-containing components with DAC by covalent bonds. Excess oxidation is irrational. It may seem that a further increase in the degree of oxidation and the number of hemostatic components will lead to a further reduction in the time to stop bleeding. However, there is a serious limitation on the oxidation state of DAC.

With an increase in the oxidation state of dialdehyde cellulose (DAC) and its protein derivatives, the rate of their hydrolytic destruction increases. At the same time, proteins diffuse into the aqueous medium not in the form of native substances, but in the form of conjugates with DAC oligomers, which makes them more resistant to inactivating factors (heat, pH of the medium, inhibitors) compared to native biologically active substances (BAS), etc. e. exhibit a prolonged specific effect. The kinetics of hydrolytic destruction is described by a semi-logarithmic anamorphosis and in logarithmic coordinates it is a straight line, the slope of which quantitatively expresses the rate constant of hydrolytic destruction, as the rate constant of the first order reaction.

Knowing these constants, it is easy to calculate the half-life of the drug by the formula

K = 0.69 / T / 2, where

K is the rate constant of destruction,

T / 2 - half-life of the drug.

Other things being equal, the degree and rate of hydrolytic destruction increases sharply with increasing pH of the medium.

Based on the foregoing, the following signs of novelty of a product can be distinguished and designated:

1. With an increase in the oxidation state of DAC, its mechanical strength sharply decreases. Data on this are shown in table 2.

table 2 The influence of the degree of oxidation of medical gauze to DAC on the strength characteristics of the material The degree of oxidation,% Breaking load (on the basis), daN Sustaining breaking load,% - 33,4 100.0 3.0 22.0 65,2 5,0 21.6 64.6 8.0 20.5 61,4 12.0 18.3 56.6 15.0 16,2 49.0 26.0 45.0 45.0

An increase in the degree of oxidation of more than 12% causes a decrease in the mechanical strength of DAC by more than 50%.

2. For the complete covalent binding of amino-containing hemostatic and bactericidal components, the degree of oxidation of DAC, selected experimentally, is necessary. An increase in the degree of oxidation, in excess of the necessary, does not lead to an increase in the hemostatic effect, but leads to an increase in the rate constant of hydrolytic degradation, which can adversely affect the mechanical strength of the material and the rate of its resorption, and hence the increased load on the protective systems responsible for removing the degradation products out of the body.

3. Immobilization per 1 g of DAC with an oxidation state of 12% of the selected hemostatic and bactericidal agents in the following amounts:

ε - aminocaproic acid - 50 mg / g

lysozyme - 5 mg / g

provides a high speed to stop bleeding and long-term preservation of the sterility of the material.

4. After the manufacture of the woven fabric, i.e. after its oxidation and immobilization of hemostatic components on it on special equipment (superfine grinding mill) DAC microfibers (powder) with particle sizes from 20 to 50 microns.

5. The time of hydrolytic destruction of the thus obtained powdery hemostatic agent is not more than 10 days.

For the first time in the world, the DAC system with an oxidation state of 12% with drugs co-immobilized on it itself receives these properties.

The time to stop bleeding is no more than 45 seconds.

Therefore, the proposed technical solution meets all the requirements for the invention: novelty, utility and prior art. The novelty consists, firstly, in the use of DAC with an oxidation state of 12% to immobilize hemostatic agents as a carrier. Secondly, ε-aminocaproic acid (without gelatin) is used as a specific hemostatic agent.

Usefulness is obvious: the rate of stopping bleeding is reduced by 25% compared with the Russian patent, which exceeds the best foreign samples in its indicators.

And finally, the prior art. The status of the medicinal product, a pharmacopoeial article and other documents are required for it to be included in the pharmacopeia of the Russian Federation.

The invention is illustrated by examples of its implementation.

Example 1

3.3 liters of distilled water are poured into the reactor, a stirrer is turned on and 280 g of iodic acid are added. The same amount of distilled water is poured into the second reactor and 46.7 g of sodium hydroxide are added with stirring. Both solutions are mixed until the acid and alkali are completely dissolved for 5-15 minutes. Then, the alkali solution is poured with stirring into the acid solution for 3-6 minutes. In the resulting sodium periodate solution, 1 kg of medical cotton gauze is placed in the form of a web and kept at room temperature in the dark for 14 hours. After activation, the web is squeezed, washed 4 times with 10 l of distilled water, squeezed again and dried in air in the dark to a residual moisture content of not more than 10% at room temperature. The gauze oxidation state is 12%.

Example 2

150 g of ε-aminocaproic acid, 15 g of lysozyme are dissolved in 6.6 L of distilled water and placed in a solution of 1 kg of DAC with an oxidation state of 12%, incubated for 3 hours at room temperature in the dark, squeezed out, dried in air in the dark to residual moisture not more than 10% and crushed.

Under experimental conditions, adult rabbits caused bleeding with the intravenous administration of 500 IU of heparin and 250 IU of fibrinolysin. Mechanically, bleeding stopped after 20-30 minutes.

Under thiopental anesthesia, superficial excision of a 1.4 cm ² liver fragment caused bleeding, a hemostatic napkin was applied to the wound, noting that the bleeding stopped after 45 seconds.

Claims (4)

1. A biodegradable hemostatic drug for stopping capillary and parenchymal bleeding in the form of microfibers, consisting of dialdehyde cellulose with blood coagulation factor epsilonaminocaproic acid and bacteriolytic enzyme lysozyme, characterized in that it contains partially oxidized cellulose di-cellulose 12 % 2. The biodegradable hemostatic drug according to claim 1, characterized in that the particle sizes of its microfiber form of partially oxidized cellulose with components immobilized on it are in the range from 20 to 50 microns. 3. Biodegradable hemostatic drug according to claim 1, characterized in that the time of complete hydrolytic destruction of complete biodegradation is not more than 10 days. 4. The biodegradable hemostatic drug according to claim 1, characterized in that the time to stop bleeding with it is not more than 45 seconds.