RU2618896C1 - Haemostatic sponge and method for its preparation - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention is a haemostatic sponge containing a base and an active substance, freeze-dried. According to the invention, the sponge, contains chitosan in acetic acid as the base and a fibrin monomer as the active substance, the components in the spongeare at a certain ratio in % by 1 litre volume.

EFFECT: expansion of haemostatic sponges assortment based on a chitosan and fibrin-monomer complex with pronounced haemostatic action.

4 cl, 13 ex

Description

The invention relates to medicine, namely to the creation of new hemostatic agents, and can be used to stop bleeding in surgical practice, in combat, in the aftermath of mass disasters and terrorist attacks, as well as in combat.

The development of military science and technology is inevitably accompanied by the emergence of new types of lesions, an increase in the severity of the pathological process, including hemorrhagic complications during gunshot wounds, and the effects of nuclear and chemical weapons. Under these conditions, a special role is played by the development of hemostatic agents to assist in critical and emergency conditions in the conditions of military operations and in disaster medicine. Prevention and stopping of bleeding are also of great importance in various fields of clinical medicine, in hematology, surgery, traumatology, oncology, obstetrics and others in connection with the introduction of new anticoagulants without antidotes into medicine, as well as drugs that have, along with therapeutic effects hepatotoxic effect. In view of the above, the creation of a new hemostatic agent that does not increase the thrombogenic potential of the blood, but which has a prophylactic and therapeutic hemostatic effect against the background of trauma and the use of antithrombotic drugs, is extremely important.

To date, there is known information about the use in surgery of various adhesive substances with hemostatic components such as fibrinogen, thrombin and some other coagulation factors (Perelman MI and other Modern adhesive compositions in thoracic surgery // Surgery. - 2002. - No. 2. - p. 47-49). Fibrin adhesives, for example, Beriplast (TM), consist of 3-4 components and must be applied to the wound surface in stages in several stages, which creates certain inconveniences during laparoscopic operations. Direct mixing of the components before use inevitably leads to the formation of a fibrin clot, loss of adhesive properties and clogging of the laparoscope lumen.

Known hemostatic sponge containing a matrix of biomaterial and one hydrophilic polymer component containing electrophilic reactive groups, and the hydrophilic component is a hydrophilic cross-crosslinker, while they bind to each other so that the reactivity of the polymer component is preserved, while " bonded "means that the specified polymer component is applied to the surface of the specified matrix of biomaterial, or the specified matrix is impregnated with the specified polymer material rial, or both, the biomaterial being selected from the group consisting of collagen, gelatin, fibrin, a polysaccharide such as chitosan, a synthetic biodegradable biomaterial such as polylactic acid or polyglycolic acid, and their derivatives (RU 2562569, 10.09. 2015). The sponge exhibits low swelling after application to the wound. However, despite the fact that the sponge is obtained by drying in air or lyophilization in a dry form, however, the water content in it is at least 0.5%. And the hemostatic properties of blood coagulation for 2 minutes are shown on a sponge sample containing only collagen as a base.

The closest and therefore taken as the closest analogue is a hemostatic sponge, which is a freeze-dried 1% solution of collagen in ammonium carbonate with a pH of 8.5-8.9, which contains feracryl, with the following ratios of components,%: collagen 98.0- 99.2, feracryl - 0.8-2.0. In this case, the sponge can be additionally structured in pairs of aldehydes and may additionally contain up to 3% antiseptics and 10% broad-spectrum antibiotics. Moreover, it has a high hemostatic activity (RU 2385726, 04/10/2010). The disadvantage of this hemostatic sponge is its adhesion to gloves and other surgical instruments.

SUMMARY OF THE INVENTION

The technical result of the claimed hemostatic sponge and method for its production is to expand the range of hemostatic sponges based on chitosan and fibrin monomer with a pronounced hemostatic effect.

The technical result is achieved by creating a hemostatic sponge containing a base and an active substance dried by freeze-drying, while it contains chitosan in acetic acid as a base, and fibrin monomer as an active substance, in the following ratio of components in a final aqueous solution in volume of 1 liter in%:

Chitosan 0.1-8.0 Acetic acid 0.1-5.0 Fibrin monomer 0.01-0.5 Water rest

In a preferred embodiment, the hemostatic sponge further comprises in the final aqueous solution in a volume of 1 liter in%: epsilon-aminocaproic acid - 0.5-10; thrombin - 0.0001-0.03 (from 1 unit of NIH to 9000 units of NIH).

Also, a method for producing a hemostatic sponge has been developed, including freeze-drying a mixture of solutions of substances, while chitosan and acetic acid are introduced into distilled water in the following ratio of components in a final concentration: chitosan from 0.1% to 8%, acetic acid from 0.1 % to 5%, dissolve chitosan with stirring and add fibrin monomer in an amount of from 0.01% to 0.5%, the resulting mixture is stirred until homogeneous and subjected to freeze-drying.

In a preferred embodiment, in addition to the mixture, from 0.5% to 10% epsilon-aminocaproic acid and from 0.0001% to 0.033% thrombin with respect to chitosan are added.

In order to give the sponge antiseptic and antibacterial effects, antiseptics (up to 3%) and broad-spectrum antibiotics (up to 10%) of the total weight of the mixture can be added to the base solution.

Fibrinogen is a large molecule that consists of three pairs of polypeptide chains combined into three domains. Thrombin hydrolyzes the Arg-Gly bonds so that two peptides A and two peptides B are cleaved from each fibrinogen molecule. A fibrin monomer forms, which tends to spontaneously polymerize into multimolecular aggregates. Fibrin monomers attached to the wound surface form primary dimers - protofibrils, in which the monomers are connected end to middle. Next, self-assembly of protofibrils occurs, the formation of bundles of fibers and stabilization using isopeptide bonds between the residues of glutamic acid and lysine. Such stabilized fibrin is the structural basis of a blood clot.

Epsilon aminocaproic acid is an inhibitor of fibrinolysis. It inhibits the activators of profibrinolysin and inhibits its conversion to fibrinolysin. It inhibits the activating effect of streptokinase, urokinase and tissue kinases on fibrinolysis. Also ε aminocaproic acid stimulates the formation of platelets, sensitizes platelet receptors to thrombin, thromboxane A2 and other endogenous aggregates.

The fibrinogen activator - thrombin - belongs to the serine proteinase family and launches a cascade of biochemical reactions of the blood coagulation system. In addition to converting fibrinogen into fibrin, it is also a powerful activator of platelet aggregation and adhesion. When thrombin acts on platelets, 5 seconds after stimulation, the cell shape changes, the granules become centralized, their contents are secreted into the open channel system and further into the extracellular medium. Due to the powerful action and danger of thrombosis, thrombin is used only locally.

Chitosan is a high molecular weight glucosamine polymer soluble in dilute organic acids. The mechanism of hemostatic action of chitosan is associated with the ability of positively charged molecules to bind to negatively charged membranes of red blood cells and form a blood clot.

Fibrin monomer is obtained according to the method described in patent RU No. 2522237. To do this, take 30 l of freshly frozen human plasma and thaw at + 37 ° C, add 10 l of a saturated solution of ammonium sulfate to the plasma with constant stirring, and the resulting mixture is kept for 15 minutes at room temperature, then centrifuged at 2500 rpm for 15 min, the supernatant is drained, then 1.4 kg of urea are dissolved in 7 L of 0.05 M phosphate buffer and heated to + 37 ° C, then the previously obtained fibrinogen precipitate is dissolved in this buffer, then 50,000 units are added here. human thrombin, stirred and the mixture was left for 1 hour at room temperature, after which the resulting mixture was divided into three parts, 40 l of 0.05 M phosphate buffer with a temperature of + 37 ° C were added to the container with one of the three parts of the initial fibrinogen solution, which formed in the container, the fibrin clot is collected, washed in distilled water and squeezed, as a result, three washed fibrin clots are obtained, then washing the fibrin clot is repeated two more times in the same way, the final product, the fibrin monomer, is obtained by dissolving the washed fibrin clot in an acetate buffer with urea, the resulting fibrin monomer is dispensed into vials at the rate of 10-20 mg / vial, then the vials with fibrin monomer is frozen and freeze-dried.

In the experiment, it was shown that a sponge made of chitosan, a fibrin monomer and with the additional administration of epsilon aminocaproic acid and thrombin has the ability to continuously reduce the time to stop bleeding and the amount of blood loss.

Examples

Example 1

Obtaining a sponge containing chitosan and fibrin monomer. 1.0 g of chitosan, 1.0 g of acetic acid are added to 500 ml of distilled water, dissolved with stirring on a shaker for 60 minutes, then a solution of 500 ml of distilled water and 0.1 g of fibrin monomer is prepared. The resulting solutions are mixed and stirred until a homogeneous mass is obtained, then poured into trays with a layer of 2 to 10 mm thick, which are placed in a freeze-drying chamber at t to -50 ° C for 48 hours before the formation of a sponge.

The ratio of components in 1 liter of the final solution: acetic acid - 0.1%, chitosan - 0.1%, fibrin monomer - 0.01%.

Example 2

Obtaining a sponge containing chitosan and fibrin monomer. 20.0 g of acetic acid and 30.0 g of chitosan are added to 500 ml of distilled water, dissolved with stirring on a shaker for 60 minutes, then a solution of 500 ml of distilled water and 2.5 g of fibrin monomer is prepared. The resulting solutions are mixed and stirred until a homogeneous mass is obtained, then poured into trays with a layer of 2 to 10 mm thick, which are placed in a freeze-drying chamber at t to -50 ° C for 48 hours before the formation of a sponge.

The ratio of components in 1 liter of the final solution: acetic acid - 2.0%, chitosan - 3.0%, fibrin monomer - 0.25%.

Example 3

Obtaining a sponge containing chitosan and fibrin monomer. 50.0 g of acetic acid and 80.0 g of chitosan are added to 500 ml of distilled water, dissolved with stirring on a shaker for 60 minutes, then a solution of 500 ml of distilled water and 5.0 g of fibrin monomer is prepared. The resulting solutions are mixed and stirred until a homogeneous mass is obtained, then poured into trays with a layer of 2 to 10 mm thick, which are placed in a freeze-drying chamber at t to -50 ° C for 48 hours before the formation of a sponge.

The ratio of components in 1 liter of the final solution: acetic acid - 5.0%, chitosan - 8.0% fibrin monomer - 0.5%.

Example 4

Obtaining a sponge containing chitosan, fibrin monomer, ε aminocaproic acid and thrombin. 1.0 g of chitosan, 1.0 g of acetic acid are added to 500 ml of distilled water, dissolved with stirring on a shaker for 60 minutes, then a solution of 500.0 ml of distilled water and 0.1 g of fibrin monomer is prepared. The resulting solutions were mixed, then 5 g of ε aminocaproic acid and 0.001 g of thrombin with an activity of 30 units were added. NIH, stirred until a homogeneous mass, then poured into trays with a layer thickness of 2 to 10 mm, which are placed in a freeze-drying chamber at t to -50 ° C for 48 hours before the formation of a sponge.

The ratio of components in 1 liter of the final solution: acetic acid 0.1%, chitosan 0.1%, fibrin monomer 0.01%, ε aminocaproic acid 0.5%, thrombin 0.0001%.

Example 5

Obtaining a sponge containing chitosan, fibrin monomer, ε aminocaproic acid and thrombin. 20.0 g of acetic acid and 30.0 g of chitosan are added to 500 ml of distilled water, dissolved with stirring on a shaker for 60 minutes, then a solution of 500.0 ml of distilled water and 2.5 g of fibrin monomer is prepared. The resulting solutions are mixed, then 50 g of ε aminocaproic acid and 0.15 g of thrombin with an activity of 4500 units are added. NIH, stirred until a homogeneous mass, then poured into trays with a layer thickness of 2 to 10 mm, which are placed in a freeze-drying chamber at t to -50 ° C for 48 hours before the formation of a sponge.

The ratio of components in 1 liter of the final solution: acetic acid - 2.0%, chitosan - 3.0%, fibrin monomer - 0.25%, ε aminocaproic acid - 5.0%, thrombin - 0.015%.

Example 6

Obtaining a sponge containing chitosan, fibrin monomer, ε aminocaproic acid and thrombin. 50.0 g of acetic acid and 80.0 g of chitosan are added to 500 ml of distilled water, dissolved with stirring on a shaker for 60 minutes, then a solution of 500.0 ml of distilled water and 50.0 g of fibrin monomer is prepared. The resulting solutions are mixed, then 100 g ε of aminocaproic acid and 0.30 g of thrombin with an activity of 9000 units are added. NIH, stirred until a homogeneous mass, then poured into trays with a layer thickness of 2 to 10 mm, which are placed in a freeze-drying chamber at t to -50 ° C for 48 hours before the formation of a sponge.

The ratio of components in 1 liter of the final solution: acetic acid - 5.0%, chitosan - 8.0% fibrin monomer - 0.5%, ε aminocaproic acid - 10.0%, thrombin - 0.03%.

Example 7

The effect of a hemostatic sponge on stopping bleeding was studied in laboratory conditions on chinchilla rabbits of both sexes weighing 3.0-4.5 kg with an average bleeding rate of 1 g / min according to the method described in the Guidelines for conducting preclinical studies of drugs ". Part one. - M .: Grif and K, 2012. The experiment was carried out with the introduction of the animal into a state of thiopental anesthesia. Then a total median laparotomy was performed, and the anterior surface of the liver was removed into the resulting wound. Using a plastic stopper, a resection of the protruding part of the liver was performed with a blade. As a result, a uniformly bleeding wound was formed. In each experiment, the size and shape of the cut segment remained unchanged. For a comparative evaluation of the hemostatic properties of the test sample and control (2 × 2 cm in size), two of the above sections were simultaneously performed on the liver lobe. A gauze swab was used as a control.

Example 8

The capillary-parenchymal bleeding was stopped by applying the sponge obtained in Example 1 to the wound. The sponge adheres well to the wound, but quickly dissolves in the blood stream and is washed off by the blood stream from the wound surface. The time to stop bleeding was 394 ± 32 s. As a control, we stopped the bleeding by tightly pressing a tampon from a gauze napkin to the wound surface. The stop of bleeding occurred in the control for 408 ± 28 s.

Example 9

The capillary-parenchymal bleeding was stopped by applying the sponge obtained in Example 2 to the wound. The sponge adheres well to the wound, has high hygroscopicity and completely stops bleeding in 90 ± 13 s. As a control, we stopped the bleeding by tightly pressing a tampon from a gauze napkin to the wound surface. The stop of bleeding occurred in the control for 408 ± 28 s.

Example 10

The capillary-parenchymal bleeding was stopped by applying the sponge obtained in Example 3 to the wound. The sponge adheres well to the wound, has high hygroscopicity and completely stops bleeding in 104 ± 16 s. As a control, we stopped the bleeding by tightly pressing a tampon from a gauze napkin to the wound surface. The stop of bleeding occurred in the control for 408 ± 28 s.

Example 11

The capillary-parenchymal bleeding was stopped by applying the sponge obtained in Example 4 to the wound. The sponge adheres well to the wound, but quickly dissolves in the blood stream and is washed off by the blood stream from the wound surface. The time to stop bleeding was 258 ± 22 s. As a control, we stopped the bleeding by tightly pressing a tampon from a gauze napkin to the wound surface. The stop of bleeding occurred in the control for 362 ± 25 s.

Example 12

The capillary-parenchymal bleeding was stopped by applying the sponge obtained in Example 5 to the wound. The sponge adheres well to the wound, has high hygroscopicity and completely stops bleeding in 86 ± 7 s. As a control, we stopped the bleeding by tightly pressing a tampon from a gauze napkin to the wound surface. The stop of bleeding occurred in the control for 326 ± 25 s.

Example 13

The capillary-parenchymal bleeding was stopped by applying the sponge obtained in Example 6 to the wound. The sponge adheres well to the wound, has high hygroscopicity and completely stops bleeding in 63 ± 6 s. As a control, we stopped the bleeding by tightly pressing a tampon from a gauze napkin to the wound surface. The stop of bleeding occurred in the control for 362 ± 25 s.

The proposed hemostatic material has a high hemostatic activity when stopping capillary-parenchymal bleeding, which allows its wide use in various fields of medicine.

Claims (5)

1. A hemostatic sponge containing a base and an active substance dried by freeze-drying, characterized in that it contains chitosan in acetic acid as a base, and fibrin monomer as an active substance, in the following ratio of components in a final aqueous solution in a volume of 1 liter at %: Chitosan 0.1-8.0 Acetic acid 0.1-5.0 Fibrin monomer 0.01-0.5 Water rest 2. The hemostatic sponge according to claim 1, characterized in that it additionally contains in the final aqueous solution in a volume of 1 liter in%: epsilon-aminocaproic acid - 0.5-10.0; thrombin - 0.0001-0.03 (from 1 unit of NIH to 9000 units of NIH). 3. A method of producing a hemostatic sponge according to claim 1, including freeze-drying a mixture of solutions of substances, characterized in that chitosan and acetic acid are added to distilled water in the following ratio of components in a final concentration: chitosan from 0.1% to 8.0% , acetic acid from 0.1% to 5.0%, dissolve chitosan with stirring and add fibrin monomer in an amount of from 0.01% to 0.5%, the resulting mixture is stirred until homogeneous and subjected to freeze-drying. 4. The method according to p. 1, characterized in that in addition to the mixture add from 0.5% to 10.0% epsilon-aminocaproic acid and from 0.0001% to 0.03% thrombin with respect to the solution.