RU2833410C1 - Haemostatic agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to a haemostatic agent. Haemostatic agent is a composition of solid thrombin and native hyaluronic acid, its water-soluble salt or its/solvate salt, thrombin content in it is not less than 0.01 IU/cm² and not more than 0.06 IU/cm².

EFFECT: agent described above is biocompatible and bioresorbable, does not swell and provides a minimum period of time to achieve haemostasis.

2 cl, 7 ex

Description

The invention relates to the field of satisfying human vital needs, namely medical products and compositions that can be used to stop bleeding and heal wounds. The hemostatic agent is biocompatible and bioresorbable due to the use of hyaluronic acid, its salts and solvates together with thrombin, preferably human.

Hyaluronic acid is a polysaccharide consisting of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine units. Depending on the number of repeats, low- (up to 100-300 thousand Da) and high-molecular (from 500 thousand, sometimes from 1000 thousand Da) are distinguished. Hyaluronic acid forms salts, as well as solvates, including solvates of salts. The term "hyaluronan" is usually used to describe this variety of compounds, and the term "hyaluronate" is used for a subset of the various salts of hyaluronic acid.

Hyaluronic acid has properties similar to its ammonium, magnesium and alkali metal salts, in particular, solubility in water (Petr Snetkov, Kseniia Zakharova, Svetlana Morozkina, Roman Olekhnovich, Mayya Uspenskaya. Hyaluronic Acid: The Influence of Molecular Weight on Structural, Physical, Physico-Chemical, and Degradable Properties of Biopolymer, p. 3. doi: 10.3390/polyml2081800).

Experts also know that when a solution of a substance is dried, solvent residues ("residual moisture") are often retained in the substance obtained in the solid phase. Such substances are usually called solvates. At the same time, some substances, including hyaluronan, are very hygroscopic and can even absorb moisture from the atmospheric air.

It is known from scientific and technical literature that in the body of mammals, including humans, hyaluronic acid is mainly represented by the high-molecular form of sodium salt. At the same time, hyaluronate is constantly resorbed and synthesized in the body; up to a third of all hyaluronic acid in the human body is resorbed and synthesized daily.

It is also known from scientific and technical literature that in solutions, hyaluronan chains can form various secondary and tertiary structures, including networks that hold water molecules.

In industry, hyaluronans are obtained mainly either by isolation from biological objects (primarily, rooster combs) or by biotechnological synthesis. Industrially obtained hyaluronan is an amorphous finely dispersed white powder with particle sizes of 0.05-0.25 mm.

A component of human blood is known - the protein thrombin (Earl W. Davie, John D. Kulman. An overview of the structure and function of thrombin, doi: 10.1055/s-2006-939550). When activating blood clotting, thrombin converts fibrinogen into fibrin and activates other blood clotting factors. Fibrin forms a mesh that captures erythrocytes, which together with platelets form a thrombus, leading to stopping bleeding.

Various hemostatic agents based on polysaccharides are known, in particular chitosan, cellulose, hyaluronic acid and their derivatives. However, it should be noted that hyaluronan has the greatest biocompatibility with humans, since it forms the extracellular matrix of vertebrates, and cellulose and chitosan perform a similar function in plants and crustaceans, fungi and insects, respectively. Cellulose and chitosan are absent in the human body.

In particular, a hemostatic sponge is known, consisting of a biomaterial - a porous matrix and a material that enhances the adhesion of the matrix to the organ (US 8771258). The biomaterial of the porous matrix includes various substances, including polysaccharides, mainly chitosan. The sponge also includes an adhesive material - two cross-linked components, represented primarily by polyethylene glycols. The disadvantage of the sponge, in addition to materials foreign to humans, is relatively slow hemostasis - 2 minutes according to example 10.

Also known is a hemostatic sponge (patent US 2021/0228764), which is a composition of oxidized cellulose and a gelatin-free bioadhesive material. The disadvantage of this product is the presence of cellulose, which is foreign to the human body.

A fast-acting dry adhesive, methods for its production and application are known (WO 2008/016983). This composition consists of two cross-linking components that are cross-linked together in the presence of conditions for such a reaction with the formation of a porous material, and a component that forms a hydrogel. Polyethyleneglycol is used as the cross-linking components. This agent was used together with thrombin in various concentrations (example 23), but no advantages of adding thrombin were found. A disadvantage of this agent is a relatively long stop of bleeding (from 1 minute in example 22 to 2 minutes in example 26).

A hemostatic, antiseptic and wound-healing sponge is known (patent RU 2226406). The sponge is made of denatured collagen - gelatin, absorbs blood 45-50 times greater than its own weight. The disadvantage of this product is its swelling, which limits its use near nerves and vessels, as well as a long (2-8 minutes) period of stopping bleeding.

A porous polymeric material based on an ionic polysaccharide is known (patent RU 2762729), which can be used as a coating for wounds and burns. This porous polymeric material is prepared from alginates, pectins, carrageenans, chondroitin sulfates or chitosan and its derivatives. Hyaluronan is not used as a base for the material, its hemostatic effect is unknown. At the same time, according to paragraph 2 of the formula of the invention, the material is converted into a water-insoluble form.

A micro-nanostructured bioplastic material is known (patent RU 2481127). Its basis is a nanostructured matrix formed from hyaluronic acid, which contains proteoglycans, glycoproteins, fibrillar proteins and an antiseptic. The material is used to treat burns, its hemostatic effect is unknown.

A method for obtaining modified hyaluronic acid is known (patent RU 2191782). The resulting chemically modified film promotes active tissue regeneration, prevents inflammatory and adhesive processes. Its hemostatic effect is unknown.

A biomaterial for preventing postoperative adhesions is known, including derivatives of hyaluronic acid (patent RU 2177332), namely its benzyl ether. The hemostatic effect of this material is unknown.

A wound dressing with a hemostatic effect is known (patent RU 2624242). This dressing consists of a biopolymer material based on bacterial cellulose and biologically active components (up to 10% hemostatic and up to 3% antimicrobial). The hemostatic agent has a fairly good speed of action. The disadvantage of the agent is that it uses cellulose, which is foreign to the human body, and has a fairly high consumption of a valuable component - thrombin "extracted" from a person (from 0.5 to 150 ME/cm ² ).

A hemostatic composition is known that includes hyaluronic acid (patent RU 2486921). This composition includes gelatin and at least 10% of native (without the use of chemical cross-linking agents) hyaluronic acid or its derivatives. The composition is stabilized by dry heat at a temperature of 110°C to 200°C. The advantage of the composition is its lower swelling, the disadvantage is relatively slow hemostasis, which takes 2 minutes or more.

A carrier with solid fibrinogen and solid thrombin is known (patent US 7399483, prototype). This carrier has similar features to the invention, namely: 1) it is used for hemostasis; 2) hyaluronic acid, among others, can be used as the carrier base (in the implementation of the invention, however, its use is not disclosed: only sponges of lyophilized and foamed collagen, gelatin, oxidized cellulose and polyglactin/dioxanone were used); 3) the carrier is coated with thrombin (1.5-2.5 IU/cm ² ) (as well as fibrinogen in an amount of 4.3-6.7 mg/cm ² ). The disadvantage of this agent is the relatively high consumption of thrombin and the use of fibrinogen - valuable resources "extracted" from humans, and also, as is known from medical literature (V.A. Gorsky, A.M. Zryanin, M.A. Agapov. Efficiency of using TachoComb in hepatobiliary surgery // Modern technologies in medicine. - 2011, No. 2, pp. 61-68), the relatively long time for hemostasis to occur - about 3 minutes.

The technical objective of the invention is to expand the technical arsenal of hemostatic agents and reduce the time required to achieve hemostasis.

The technical result of the invention consists in the creation of a new hemostatic agent based on hyaluronan and thrombin, which is biocompatible and bioresorbable, does not swell and ensures a minimum period of achieving hemostasis.

Experiments were previously conducted with various forms of hyaluronan and thrombin concentrations, which unexpectedly showed that the said technical result is achieved in the case where the hemostatic agent is a composition of native hyaluronan (namely, hyaluronic acid, its water-soluble salt, or its/salt solvate) and thrombin, which are in solid form, wherein the thrombin content is at least 0.01 ME per cm ² . In some embodiments of the invention, hyaluronan may be in the form of at least a partially crystallized polymer. The specified result is achieved due to the fact that hyaluronan binds water in blood plasma, which increases the concentration of components in blood plasma and ensures more efficient and rapid platelet adhesion, and thrombin contained in the agent converts native fibrinogen into fibrin, which forms fibrin networks additional to hyaluronan networks, immobilizing erythrocytes, resulting in the formation of a clot (thrombus) that stops bleeding, while the formation of this thrombus and the stopping of bleeding are faster, the more reactive the hyaluronan is and the higher the concentration of thrombin in the agent. To impart additional properties or accelerate the speed of action, the hemostatic agent may include other blood clotting factors, cell growth factors, antiseptics, anesthetics and/or wound healing substances (e.g. methyluracil, dexpanthenol). The product may also use auxiliary substances that do not determine its key functionality, such as, for example, riboflavin, silicone to distinguish the "active" and reverse surfaces and to increase the convenience of applying the product to the wound. The product may be sterilized by methods known to a specialist, for example, ethylene oxide.

The proposed technical solution is new and is not described in patent and scientific literature.

The proposed hemostatic agent can be used, for example, to stop bleeding and accelerate wound healing.

The content of the invention, differences and advantages of the proposed hemostatic agent are illustrated by the following examples.

Example 1

Sodium hyaluronate with a molecular weight of 11.6*10 ⁵ , produced by the biotechnological method of Swedlight AB (Sweden), was dissolved in 10 g in 2 l of distilled water and poured onto a tray with an area of 1478 cm ² . Then, a solution of thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Hemophilia Patients) with an activity of 9 ME was added to it, evenly distributing it over the surface. The resulting solution was frozen on a tray to a temperature of -60 ° C and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to + 25 ° C with increasing pressure for 96 hours, while at the last stage of drying, the maximum possible vacuum for the lyophilization apparatus was again created. A homogeneous white non-woven fabric was obtained, which was cut into 3×4 cm samples. The thrombin concentration in the composition was 0.006 ME/cm ² .

Example 2

Sodium hyaluronate with a molecular weight of 11.6*10 ⁵ , produced by the biotechnological method of Swedlight AB (Sweden), was dissolved in 10 g in 2 l of distilled water and poured onto a tray with an area of 1478 cm ² . Then, a solution of thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Hemophilia Patients) with an activity of 18 IU was added to it, evenly distributing it over the surface. The resulting solution was frozen on a tray to a temperature of -60 ° C and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to + 25 ° C with increasing pressure for 96 hours, while at the last stage of drying, the maximum possible vacuum for the lyophilization apparatus was again created. A homogeneous white non-woven fabric was obtained, which was cut into 3×4 cm samples. The thrombin concentration in the composition was 0.01 ME/cm ² .

Example 3

Sodium hyaluronate with a molecular weight of 11.6*10 ⁵ , produced by the biotechnological method of Swedlight AB (Sweden), was dissolved in 10 g in 2 l of distilled water and poured onto a tray with an area of 1478 cm ² . Then, a solution of thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Hemophilia Patients) with an activity of 90 IU was added to it, evenly distributing it over the surface. The resulting solution was frozen on a tray to a temperature of -60 ° C and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to + 25 ° C with increasing pressure for 96 hours, while at the last stage of drying, the maximum possible vacuum for the lyophilization apparatus was again created. A homogeneous white non-woven fabric was obtained, which was cut into 3×4 cm samples. The thrombin concentration in the composition was 0.06 ME/cm ² .

Example 4

An experiment was conducted on adult female rabbits. For the experiment, a liver wound was modeled: first, a median laparotomy was performed with the liver edge exposed, then the parenchyma with the liver capsule was dissected, and the compositions from examples 1-3 were applied to the wound.

The composition from example 1 dissolved almost immediately without visible changes in the wound; after 3 minutes, hemostasis was not observed.

The composition from example 2 dissolved in 15 seconds, the onset of thrombus formation was observed, which ended with hemostasis after 1.5 minutes.

The composition from example 3 did not dissolve completely; hemostasis occurred within 15 seconds.

Example 5

Sodium hyaluronate with a molecular weight of 11.6*10 ⁵ , produced by the biotechnological method of Swedlight AB (Sweden), weighed 10 g and 0.2 g of benzyldimethyl[3-(myristoylamino)propyl]ammonium chloride monohydrate were dissolved in 2 l of distilled water and poured onto a tray with an area of 1478 cm ² . Then, a solution of thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Hemophilia Patients) with an activity of 18 ME was added to it, evenly distributing it over the surface. The resulting solution was frozen on a tray to a temperature of -60°C and dried in a lyophilic drying apparatus by stepwise increasing the temperature from the freezing temperature to +25°C with increasing pressure for 96 hours, while at the last stage of drying the maximum possible vacuum for the lyophilic apparatus was again created. A homogeneous white non-woven fabric was obtained.

The obtained sample was used to study the antimicrobial effect in accordance with OFS 1.2.4.0002.18 (source: State Pharmacopoeia of the Russian Federation, IV edition. Volume 1. Methods of analysis of medicinal products. Methods of biological analysis. Microbiological purity, p. 1128).

We used test strains of microorganisms representing various taxonomic groups of bacteria and a test strain of yeast-like fungi: Pseudomonas aeruginosa and Escherichia coli, representatives of Gram-negative bacteria; Bacillus cereus, a representative of Gram-positive spore-forming bacteria; Staphylococcus aureus, a representative of Gram-positive bacteria; Candida albicans, a representative of yeast-like fungi.

As a result of the growth test, none of the microorganisms were detected.

Example 6

Sodium hyaluronate with a molecular weight of 11.6*10 ⁵ , produced by a biotechnological method by the company Swedlight AB (Sweden), in a weighed amount of 10 g and 0.2 g of benzyldimethyl[3-(myristoylamino)propyl]ammonium chloride monohydrate were dissolved in 2 l of distilled water and poured onto a tray with an area of 1478 cm ² in 3 repetitions.

Next, a solution of thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Hemophilia Patients) with an activity of 9, 18 and 90 ME, respectively, was added to each tray, evenly distributing it over the surface. The resulting solutions were frozen on the tray to a temperature of -60°C and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to +25°C with increasing pressure for 96 hours, while at the last stage of drying, the maximum possible vacuum for the lyophilization apparatus was again created. Homogeneous white nonwoven fabrics were obtained, which were cut into 3x4 cm samples.

The obtained samples were then tested in an experiment on a thigh wound model. Adult female rabbits were used for the experiment. To create a thigh muscle wound, the skin, subcutaneous fat, fascia and the muscle itself (longitudinally, to simulate severe bleeding) were dissected. After the applications, the wound was left open for 3 days.

Based on the results of the experiment:

- a composition with a thrombin activity of 0.006 IU/ ^cm2 - the application completely dissolved, hemostasis was not observed for more than 3 minutes, after 3 days exudation with signs of inflammation was detected in the wound;

- a composition with a thrombin activity of 0.01 IU/ ^cm2 - the application completely dissolved, hemostasis occurred after 2 minutes, after 3 days minimal exudation with limited signs of inflammation was detected in the wound;

- a composition with a thrombin activity of 0.06 IU/ ^cm2 - hemostasis occurred after 20 seconds, after 3 days the wound is completely dry, there is no exudation or inflammation, there are remnants of the incompletely dissolved application in the wound.

Example 7

Sodium hyaluronate with a molecular weight of 11.6*10 ⁵ , produced by the biotechnological method of Swedlight AB (Sweden), weighed 10 g, and 0.2 g of benzyldimethyl[3-(myristoylamino)propyl]ammonium chloride monohydrate, and 20 g of lidocaine were dissolved in 2 l of distilled water and poured onto a tray with an area of 1478 cm ² . Then, a solution of thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Hemophilia Patients) with an activity of 90 ME was added to it, evenly distributing it over the surface. The resulting solution was frozen on a tray to a temperature of -60°C and dried in a lyophilic drying apparatus by stepwise increasing the temperature from the freezing temperature to +25°C with increasing pressure for 96 hours, while at the last stage of drying the maximum possible vacuum for the lyophilic apparatus was again created. A homogeneous white non-woven fabric was obtained. Samples of 1×1 cm were cut from this fabric.

These samples were applied by placing them in the socket of an extracted tooth to stop bleeding in two adult male patients after extraction of teeth 38 and 48. The bleeding in the patients stopped within 10 and 15 seconds. When re-administered 3 days later, the wound healed without inflammation, and the patients did not require additional anesthesia.

Thus, from the given examples it is clear that the proposed hemostatic agent has a high speed of action and efficiency and can be used in various conditions, including to stop bleeding in infected wounds or to prevent their infection.

Claims (2)

1. A hemostatic agent, which is a composition of thrombin and native hyaluronic acid, its water-soluble salt or its/salt solvate, in solid form, characterized in that the thrombin content in it is not less than 0.01 IU/ ^cm2 and not more than 0.06 IU/ ^cm2 . 2. A hemostatic agent according to claim 1, characterized in that the native hyaluronic acid, its water-soluble salt or its/salt solvate is partially in crystalline form.