WO2025144087A1 - Haemostatic agent - Google Patents

Abstract

The invention relates to the field of materials science and medicine and concerns a novel haemostatic agent based on hyaluronan which can be used, in particular, in medicine for stopping bleeding and for healing wounds. The proposed haemostatic agent very quickly stops bleeding and exhibits good adhesion to body tissues. This technical result is achieved in that the haemostatic agent is a composition comprised of thrombin and hyaluronan, which are provided in solid form, and is characterized by a thrombin content of not less than 0.01 IU/cm2 and not more than 109 IU/cm2. The haemostatic agent may further contain an antiseptic component and/or an analgesic component and/or a component that promotes wound healing.

Description

Hemostatic agent

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 hyaluronan - 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-O-glucosamine units. Depending on the number of repeats, low- (up to 100-300 thousand Da) and high-molecular (from 500 thousand, sometimes from 1,000 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 commonly called solvates. At the same time, some substances, including hyaluronan, are highly 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, hyaluronan is constantly resorbed and synthesized in the body; up to a third of all hyaluronan 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 red blood cells, 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 8 771 258). The biomaterial of the porous matrix includes various substances, including polysaccharides, primarily chitosan. The sponge also includes an adhesive material - two cross-linked components, represented primarily by polyethylene glycols. A 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 glue, 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. Polyethylene glycol is used as the cross-linking components. This agent was used together with thrombin in various concentrations (example 23), but no advantages were found in adding thrombin. The disadvantage of this agent is a relatively long stop of bleeding (from 1 minute in example 22 to 2 minutes in example 26), as well as the use of a substance not found in the human body - polyethylene glycol. A hemostatic, antiseptic and wound-healing sponge is known (patent RU 2 226 406). The sponge is made of denatured collagen - gelatin, absorbs blood 45-50 times its own weight. The disadvantage of this product is its swelling, which limits its use near nerves and vessels, since it can lead to compression of the vessel or nerve with the corresponding negative consequences, 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 2 762 729), 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. In this case, 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 2 481 127). 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 2 191 782). The resulting chemically modified film promotes active tissue regeneration, prevents inflammatory and adhesive processes, but has reduced biodegradability. Its hemostatic effect is unknown. A biomaterial for preventing postoperative adhesions is known, including derivatives of hyaluronic acid (patent RU 2 177 332), namely its benzyl ether. The hemostatic effect of this material is unknown.

A wound dressing with a hemostatic effect is known (patent RU 2 624 242). 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 / square centimeter).

A hemostatic composition is known that includes hyaluronic acid (patent RU 2 486 921). 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 degrees Celsius to 200 degrees Celsius. 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 7,399,483). 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 base of the carrier (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 ME / square centimeter) (and also fibrinogen in the amount of 4.3-6.7 mt / square centimeter). 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), a relatively long period of hemostasis - about 3 minutes.

A “molded sheet product” is known, consisting of various compositions of proteins, aliphatic polyesters and water-soluble polymers, some of the variants of which have a haemostatic effect (EP 2851095).

The hemostatic effect, as shown in the description of the said invention, is not possessed by any of these materials (in particular, the combination of a water-soluble polymer - hyaluronan - and thrombin claimed in the present invention), but by a combination of these materials - sheets of a water-soluble polymer with fibrinogen and an aliphatic polyester with thrombin superimposed on each other (example 21) or the same sheets pre-laminated with each other (examples 24, 27, 29, 31). Also claimed in example 30 is a hemostatic effect (manifested after 3 minutes) of a composition of thrombin (in an amount of 24.2 ME / square centimeter) and an aliphatic polyester.

In this case, no examples of obtaining a water-soluble polymer composition with thrombin are given. The source also states (p. 12 of the description, first paragraph) that the supporting characteristics of the product formed by the proposed method are excellent, in contrast to the product obtained by the lyophilization method. The hemostatic material declared in the present invention also has excellent supporting characteristics, and was obtained by lyophilization, which emphasizes that the materials proposed by the applicant and in the specified document differ.

Thus, the hemostatic material claimed in the present invention and the combination of molded sheet materials having a hemostatic effect according to patent EP 2851095 differ significantly in composition.

The claims of patent EP 2851095 do not claim a hemostatic effect or thrombin content. The description also does not provide information on the hemostatic effect of the thrombin composition (with a content of from 0.01 IU / square centimeter to 100 square centimeters) and a water-soluble polymer, for example, hyaluronan. In particular, on page 7 of the description (1st paragraph) it is indicated that when the thrombin content is less than 0.01 IU / square centimeter, there is no hemostatic effect, when the thrombin content is more than 100 IU / square centimeter, the molded sheet product becomes brittle, and the presence of a hemostatic effect in a water-soluble polymer containing thrombin in the range of 0.01-100 IU / square centimeter is not disclosed in invention EP 2851095. The hemostatic effect is indicated (in example 30) only in relation to a combination of thrombin (in an amount of 24.2 IU / square centimeter) and an aliphatic polyester. In other examples where fibrinogen is used in a water-soluble polymer in addition to thrombin in an aliphatic polyester, the thrombin content in the aliphatic polyester ranges from 14.7 IU/square centimeter (Example 20) to 31.39 IU/square centimeter (Example 15).

Thus, the hemostatic effect of a composition of a water-soluble polymer and thrombin in an amount from 0.01 IU/square centimeter to 100 IU/square centimeter is not known from the prior art (patent EP 2851095). At the same time, as is known from level of technology (Ditrich, M., Snejdrova, E. (2014). Cyclic Swelling as a Phenomenon Inherent to Biodegradable Polyesters. Journal of Pharmaceutical Sciences, 103(11), 3560-3566. doi: 10.1002/jps.24146), biodegradable aliphatic polyesters are characterized by swelling, which is undesirable, and this problem is solved by the claimed technical solution.

Also, the invention according to patent EP 2851095 claims a "molded sheet product" ("formed by laminating, weaving, knitting or processing by other methods of one or more fibers"). The need to obtain a molded product complicates the production process, which is a disadvantage, and limits the possibilities of its application (for example, when the material is required to be free-flowing). At the same time, a hemostatic material in powder form is in demand for hemostasis in hard-to-reach places where it is difficult to apply a sheet product. The claimed material does not require molding and can be presented in a wider range of finished forms, which expands the technical arsenal of hemostatic agents.

A pharmaceutical composition is known, consisting of a polymer carrier (for example, hyaluronic acid) and thrombin or fibrinogen, located in a glassy carrier, which is distributed inside or on the surface of a second - polymer - carrier (WO 2013004838A1 prototype).

The rate of onset of the hemostatic effect of the material, according to the description of the specified composition, is from 10 to 2 minutes for moderate bleeding (p. 24, paragraph 4).

An essential feature of the said composition is the presence of thrombin in a glassy carrier. The disadvantage of the prototype is relatively long period of time for hemostasis to occur, which is 120 seconds or more.

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, has adhesion to body tissues and ensures a reduction in the time to achieve 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 and thrombin in solid form, with the thrombin content being no less than 0.01 ME per square centimeter and no more than 109 ME per square centimeter. In some embodiments of the invention, hyaluronan may be in the form of at least a partially crystallized polymer. This 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 adhesion of platelets, and thrombin contained in the product 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 stopping of bleeding are faster, the more reactive is hyaluronan and the higher the concentration of thrombin in the product. To give additional properties or to accelerate the speed of action, the hemostatic agent may include other blood coagulation factors, cell growth factors, antiseptics, anesthetics and/or wound-healing substances (e.g., methyluracil, dexpanthenol). Also, the agent may 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 agent to the wound. The agent can be sterilized by methods known to a specialist, for example, ethylene oxide or radio emission.

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) in a weighed amount of 10 g was dissolved in 2 liters of distilled water and poured onto a tray with an area of 1478 square centimeters. Then, a solution of thrombin reagent PG-9 A 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 degrees Celsius and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to +25 degrees Celsius with increasing pressure for 96 hours, while at the last stage of drying the maximum possible for lyophilic apparatus vacuum. A homogeneous non-woven white fabric was obtained, which was cut into 3 x 4 cm samples. The concentration of thrombin in the composition was 0.006 ME / square centimeter.

Example 2.

Sodium hyaluronate with a molecular weight of 11.6*10 ⁵ , produced by the biotechnological method of Swedlight AB (Sweden), in a weighed amount of 10 g was dissolved in 2 l of distilled water and poured onto a tray with an area of 1478 square centimeters. 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 degrees Celsius and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to +25 degrees Celsius 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 x 4 cm samples. The thrombin concentration in the composition was 0.01 IU/square centimeter.

Example 3.

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

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 without visible changes in the wound; after 3 minutes, hemostasis was not observed.

The composition from Example 2 initially “stuck” to the wound, but subsequently mostly dissolved within 15 seconds, and thrombus formation began, which ended with hemostasis within 1.5 minutes.

The composition from example 3 firmly “stuck” to the wound, did not dissolve completely, and hemostasis occurred within 15 seconds.

Example 5.

Sodium hyaluronate with a molecular weight of 11.6* 10 ⁵ , produced by the biotechnological method of 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 liters of distilled water and poured onto a tray with an area of 1478 square centimeters. Then, evenly distributed over the surface, 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. The resulting solution was frozen on a tray to a temperature of -60 degrees Celsius and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to +25 degrees Celsius 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.

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 - representative of Gram-positive spore-forming bacteria; Staphylococcus aureus - representative of Gram-positive bacteria; Candida albicans - 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 10 g sample, 0.2 g benzyldimethyl [3-(myristoylamino)propyl] ammonium chloride monohydrate and 20 g dioxomethyltetrahydropyrimidine dissolved in 2 liters of distilled water and poured onto a tray with an area of 1478 square centimeters 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 degrees Celsius and dried in a lyophilization apparatus by stepwise increasing the temperature from the freezing temperature to +25 degrees Celsius 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 non-woven fabrics were obtained, which were cut into 3 x 4 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 ME/square centimeter - the application completely dissolved, hemostasis was not observed for more than 3 minutes, after 3 days minimal exudation with limited signs of inflammation was detected in the wound;

- a composition with a thrombin activity of 0.01 ME/square centimeter - the application completely dissolved, hemostasis occurred after 2 minutes, after 3 days there are remnants of the application that has not completely dissolved in the wound; - a composition with a thrombin activity of 0.06 ME/square centimeter - 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 the company Swedlight AB (Sweden) in a weighed amount of 10 g and 0.2 g of benzyldimethyl [3-(myristoylamino)propyl] ammonium chloride monohydrate and 20 g of lidocaine were dissolved in 2 liters of distilled water and poured onto a tray with an area of 1478 square centimeters. Then, a solution of thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Patients with Hemophilia) 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 degrees Celsius and dried in a lyophilic drying apparatus by stepwise increasing the temperature from the freezing temperature to +25 degrees Celsius 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 x 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.

Example 8. Sodium hyaluronate with a molecular weight of 11.6* 10 ⁵ , produced by the biotechnological method by Swedlight AB (Sweden) in a weighed amount of 1 g and thrombin reagent PG-9A for hemostasis studies produced by NPO Renam (MBOOI Society of Hemophilia Patients) with an activity of 16200 IU were dissolved in 0.2 l of distilled water and poured onto a tray with an area of 148 square centimeters. The resulting solution was frozen on the tray to a temperature of -60 ° C and dried in a freeze-drying apparatus by stepwise increasing the temperature from the freezing temperature to +25 degrees Celsius 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 dense, homogeneous white non-woven fabric was obtained. The concentration of thrombin in the composition was 109 IU / square centimeter. When bent, the canvas broke and partially crumbled.

A piece of material measuring approximately 1x1 cm was crushed into a Petri dish containing 6 ml of blood plasma prepared from “Control Plasma KM-2” manufactured by NPO Renam. Almost instantaneous clot formation was observed, although the material did not dissolve completely. Thus, with a thrombin content of up to 109 IU/cm2, the material retains its hemostatic properties. The material is still in solid form and can be used in powder form for, for example, laparoscopic applications.

Example 9.

The sample obtained in Example 2 was examined by X-ray phase analysis on an ARL X'TRA X-ray powder diffractometer (Figure). According to the results of X-ray phase analysis, the samples have a partially amorphous (which is characterized by a halo on the diffraction pattern), partially crystalline structure, which is characterized by the presence of peaks in the region of 24.8 and 43.8 degrees theta.

Brief description of the drawings: the figure “Diffractogram of a sample according to the invention” shows a diffractogram of a sample manufactured in example 2, obtained by X-ray phase analysis.

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

Claims

Invention formula 1. A hemostatic agent, which is a composition of thrombin and hyaluronan in solid form, characterized in that the thrombin content in it is not less than 0.01 IU/square centimeter and not more than 109 IU/square centimeter. 2. A hemostatic agent according to claim 1, characterized in that the hyaluronan is partially in crystalline form. 3. A hemostatic agent according to item 1, characterized in that it additionally contains an antiseptic and/or analgesic and/or wound-healing component.