RU2826361C1 - Haemostatic agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a local haemostatic agent. Hemostatic agent containing chitosan derivatives and hydrolysed collagen, wherein the chitosan derivatives used are its salt and additionally tannin and arginine, or oligoarginine, or polyarginine, or protamine sulphate, with the following ratio of components, wt.%: chitosan salt 2–4, tannin 1, arginine 1, or oligoarginine 1, or polyarginine 1, or protamine sulphate 1, hydrolysed collagen 20, water—the balance.

EFFECT: use of the invention provides the creation of an effective haemostatic agent with high adhesion, including to a wet wound surface, which does not require additional materials for its fixation.

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Description

The invention relates to the field of medicine, namely to local hemostatic agents, and can be used to stop external bleeding, as well as for injuries to parenchymatous organs and large vessels.

Today, local hemostatic agents are widely used not only in surgical clinics, but also at the pre-hospital stage. These drugs are convenient for intraoperative hemostasis and when providing first aid to the wounded. During surgical interventions, local hemostatic agents can reduce or prevent intraoperative blood loss. Topical hemostatics are easy to use and do not contribute to additional tissue trauma, which is possible when suturing or bandaging the source of bleeding, using diathermocoagulation, laser photocoagulation and other physical methods of hemostasis.

Modern materials used for the production of local hemostatic agents differ in their nature. According to their composition, they can be divided into the following groups:

- materials based on biological blood clotting factors obtained from animals or humans: collagen, thrombin, fibrin, fibrinogen, plasma and others;

- materials based on cellulose derivatives;

- materials based on synthetic polymers;

- materials based on inorganic compounds (zeolite, silica gel);

- materials based on plant extracts;

- materials based on chitosan, etc.

In terms of release form, there are hemostatic agents in the form of gauze with the active substance applied to it, hemostatic sponges, plates, powders, etc.

The most common local hemostatic agents are based on chitosan and collagen.

Chitosan is a natural polysaccharide isolated from crustacean shells; the positive charge of the _-NH2 group of chitosan interacts electrostatically with anions on the surface of red blood cells, which leads to intense aggregation of red blood cells around the wound with the formation of thrombi, which quickly stop bleeding. Chitosan promotes the activation of platelets and increases their adhesion. In addition, in vivo chitosan leads to contact activation of blood coagulation factors, strengthening the clot. It is biologically inert and biodegradable. Lysozyme is the main enzyme responsible for the degradation of chitosan in vivo by hydrolysis of acetylated residues. Lysozyme is also found in blood (Ovsyannikov V.G., Toropkina Yu.E., Kraskevich V.V., Alekseev V.V., Boychenko A.E., Alekseeva N.S., Kraskevich D.A. Lysozyme - the limits of the possible// Modern problems of science and education. - 2020. - No. 3). The end products of degradation are soluble chitosan oligosaccharides of short length. Chitosan has a stimulating effect on macrophages and is also a chemoattractant for neutrophils, which helps to accelerate the biodegradation of chitosan. Chitosan also has non-specific antimicrobial activity and promotes wound healing (Di Martino, A. Chitosan: a versatile biopolymer for orthopaedic tissue-engineering / A. Di Martino, M. Sittinger, MV Risbud // Biomaterials. -2005. - Vol. 26, № 30. - P. 5983-5990..), (Da Sacco, L. Chitin and chitosan as multipurpose natural polymers for groundwater arsenic removal and AS2O3 delivery in tumor therapy / L. Da Sacco, A. Masotti// Mar. Drugs. - 2010. - Vol. 8, № 5. - P. 1518-1525). All these properties make chitosan one of the most sought-after substances for the creation of local hemostatic agents and tissue bioadhesives.

Patent RU2675631C1 describes a local hemostatic agent containing chitosan, carboxymethylcellulose and ciprofloxacin. The drug is used as a first aid for external wounds, and helps prevent infection in the wound.

The bandage "HEMOFLEX" (RU248770) is created on the basis of chitosan nanofibers. It has a large specific surface, porosity, air permeability and increased sorption properties.

The preparations produced in the territory of the Russian Federation also include "GEPOGLOS" (RU2635465), produced in the form of a bandage with chitosan applied to it. The active substance - acylated chitosan salt, is applied to both sides of the bandage. The bandage also contains carboxymethyl starch to increase the sorption capacity and is made of non-woven material "spunlace" containing viscose and polyester.

The hemostatic agent "GEMOKHIT" (RU2681184C1) is known, in the production of which chitosan cross-linked with epoxy resin is used, which is due to its higher absorption capacity compared to non-cross-linked chitosan. The use of glycerin as a plasticizer (which is due to its high plasticizing properties) gives the sponge the necessary elasticity and strength.

HemCon Medical Technologies Inc. in the USA produces the following hemostatic products: "HEMCON GUARDACARE PRO" US 9205170 B2 (wound dressings containing chitosan) and "HEMCON BANDAGE PRO" (bandages with chitosan applied to them). This product is widely used in combat conditions to provide assistance to the wounded.

Known patent US 5836970 A: wound dressings containing chitosan and alginate. According to the patent, this medical product can be produced in the form of powder, film, gel, foam or their mixtures.

Among local hemostatic agents containing chitosan, patent US 20050123588 A1 is known, which presents a hemostatic agent containing chitosan fibers, onto which hemostatic microporous polysaccharide microspheres and a medicinal product or biologically active substance are applied. The hemostatic material is recommended by the authors for stopping active bleeding in wounds of arteries and veins, sealing punctures of femoral arteries.

Also known is patent US 20050203058 A1, which describes a hemostatic agent containing α- and β-chitosan and their derivatives.

Patent number US 6998509 B1 discloses a wound treatment product containing chitosan. The chitosan in this product is in the form of fibers modified by treatment with acid in a solvent that is incapable of dissolving chitosan fibers and by heat treatment.

Patent US 8106030 B2 describes an invention in which a hemostatic powder comprises a chitosan salt together with at least one inert material.

Patent GB 2393120 A describes a composite wound dressing containing chitosan and oxidized cellulose. The preparation may be in the form of a sponge or a wound dressing.

Invention CN 1833732 A is known, where carboxymethyl chitosan fibers are obtained from chitosan fibers by chemical modification. It can be used to prepare surgical dressings for wounds and burns, postoperative dressings.

Patent WO 2020021499 A1 presents an invention that describes a hemostatic gel dressing containing chitosan as a polysaccharide, gelatin as a protein and starch as a carbohydrate in one phase.

Known patent CN 105169459 B describes the following method for preparing a hemostatic powder: mixing highly viscous chitosan with a high degree of deacetylation with aldehyde acid, sodium alginate and zinc.

Patent No. CN 105251036 A presents a hemostatic material containing by weight 60-75 parts of chitosan fiber, 6-15 parts of keratin, 10-25 parts of sodium carboxymethylcellulose and 5-10 parts of polyvinyl alcohol. The medical hemostatic material has such advantages as rapid hemostasis, excellent mechanical properties, good biocompatibility, etc.

Patent KR 101699924 B1 discloses a method for producing medical fiber containing calcium carboxymethylcellulose and a chitosan compound.

Also known is patent CN 104874011 A describing a hemostatic agent, which is a solid preparation obtained by the reaction of a chitosan derivative, hyaluronic acid, chondroitin sulfate, alginic acid, carbomer, xanthan gum or a cellulose derivative and a cross-linking agent. The cross-linking agent is formaldehyde, methylglyoxal, glutaraldehyde. The hemostatic has the advantages of a fast and effective hemostatic effect, good biocompatibility and biodegradability.

Patent RU 2759898 C2 proposes an absorbent polymer hydrogel xerogel sponge, which is a porous sponge with a three-dimensional network with chitosan as its base, an absorbent polymer and a macromolecule or polymer as a cross-linking agent. The sponge can be used for emergency hemostasis of large arteriovenous bleeding.

Patent RU2589840C1 describes the invention of a hydrophilic gel for wound covering, containing chitosan succinate, water and ethyl alcohol as auxiliary substances. The technical result is a hydrophilic gel for wound covering with improved properties due to increased penetrating ability and healing and disinfection and preservation of shape on the wound.

The disadvantages of all analogues include low adhesiveness, which means that additional fixation of the drug to the wound is necessary, as well as the fact that the active substance containing chitosan or its salts loses mechanical strength upon contact with body fluids and gradually gets wet. In addition, all of the listed analogues are recommended for use only for external wounds; their use for intracavitary wounds is not described.

Collagen-based products have also proven themselves to be effective hemostatic agents.

Collagen is a protein that forms the basis of connective tissue in animal organisms. The structural and chemical stability of collagen, its physical properties are due to the unique organization of three-spiral macromolecules, which, in turn, at physiological temperature, pH and ionic strength aggregate to form a three-dimensional network of intermolecular bonds of various natures. When incoming molecules and cells come into contact with the "loops" of collagen spirals, inclusion compounds (clathrates) are formed. Collagen accelerates the natural path of coagulation and starts the blood clotting process at the moment of the first contact of the material fibrils with platelets.

Aggregated platelets undergo normal morphological changes - degranulate, releasing ADP, serotonin, thromboxane A2, which helps form a clot. Thus, the main hemostatic mechanism of collagen action is platelet aggregation and activation.

Dispersed collagen in concentrations above 0.5% forms stable emulsions of the M/B type. Under dehydrating conditions, transparent non-fragile films are obtained from a thin dispersion layer, and powders are obtained with spray drying. The technology for producing collagen sponges is based on the method of sublimation (lyophilic) drying of a thin layer of collagen solution. In the process of sublimation of frozen water crystals in a deep vacuum, highly porous sponges are obtained, in which the volume of through and dead-end pores is 50-90% of the total sponge volume. Such a sponge has a high sorption capacity and absorbs an amount of moisture that is many times greater than its own weight, practically without increasing in size. Hemostasis is achieved within 2-5 minutes. Within 5 minutes, collagen forms a water- and air-permeable gel-like layer, but the sponge plate must be pressed tightly to the wound surface (the preparation adheres poorly to an uneven wound surface due to the rigidity of the collagen fibers).

A collagen sponge is known from patent RU 2385726 C1, which additionally contains up to 10% broad-spectrum antibiotics and up to 3% antiseptics. The disadvantage of this hemostatic sponge is its adhesiveness to gloves and other surgical instruments.

Invention RU 2584348 C2 describes a composite collagen sponge, which is intended to stop bleeding, as well as to stimulate cell growth and proliferation. The sponge contains collagen, cell growth factors and a protective agent in an amount of 10%-50% by weight, where the protective agent contains amino acids, saccharides and albumin. The sponge has a water absorption capacity of more than 52 times based on the weight of the sponge and has an elastic modulus of more than 70.0 N/cm2.

The method for producing a collagen sponge described in patent EA 005697 B1 comprises adding air to a collagen gel and stirring to produce a collagen foam, which is then dried. From the dried product thus obtained, a collagen sponge is obtained by separating parts of the sponge with a chamber diameter greater than 0.75 mm and less than 4 mm or parts with an average diagonal chamber size of 3 mm. The collagen sponge can be used as a wound closure material, possibly coated with a fibrin glue, for example a combination of fibrinogen, thrombin and aprotinin.

Among the analogs that are applicable for intraoperative use when working with wounds of parenchymatous organs and vessels, it is necessary to mention preparations based on coagulation factors. They are a combination of various coagulation factors. These include, for example, "TACHOSIL" US 9517287 B2, which is a collagen sponge coated with fibrinogen and thrombin isolated from human blood. This preparation has a fast and reliable hemostatic effect.

"TACHOSIL" is a more advanced modification of the drug "TACHOCOMB" (EP0059265B1). The latter is made of horse collagen, bovine thrombin, bovine aprotinin and human fibrinogen. "TACHOSIL" contains only human fibrinogen with horse collagen as a carrier. It does not contain aprotinin or other components of bovine origin. The essence of such products is to imitate the final stages of coagulation. They ensure hemostasis in 3-5 minutes. Upon contact with a bleeding surface, the drug becomes active, releasing active coagulation factors. Their biodegradation in the body lasts about 12 weeks.

Such hemostatic agents demonstrate a fast and reliable hemostatic effect, have bioadhesiveness, and are suitable for intracavitary use. However, these drugs carry a large antigen load and also contain human blood proteins, which entails the risk of the patient becoming infected with viral infections. In addition, they are undoubtedly more expensive than their synthetic analogues based on chitosan, kaolin, etc.

It is worth noting that the effectiveness of collagen sponges is manifested only in case of moderate parenchymal bleeding. They also have poor adhesion to the wound surface and can lose contact with the wound, which can potentially lead to recurrent bleeding. In case of massive, mixed bleeding, they are “washed off” from the wound surface [Budko E.V., Chernikova D.A., Yampolsky L.M., Yatsyuk V.Ya. Local hemostatic agents and ways to improve them // Rus. med.-biol. vestn. im. acad. I.P. Pavlova. 2019. No. 2]. The use of tissue growth factors in them as wound healing agents increases the risk of allergic reactions. Also, the carcinogenic risks associated with the use of growth factors are not fully understood.

The closest to the proposed one is a biodegradable collagen sponge reinforced with chitosan/calcium pyrophosphate nanoflowers for rapid hemostasis (Yan T, Cheng F, Wei X, Huang Y, He J. Biodegradable collagen sponge reinforced with chitosan/calcium pyrophosphate nanoflowers for rapid hemostasis. Carbohydr Polym. 2017 Aug 15;170:271-280. doi: 10.1016/j.carbpol.2017.04.080. Epub 2017 Apr 29. PMID: 28521997.), which we chose as a prototype.

The authors of the prototype developed a biodegradable collagen sponge reinforced with chitosan and calcium pyrophosphate nanoparticles (CPNFs-Col sponge) for rapid hemostasis. With specific characteristics such as the ability to quickly absorb water, a positive charge on the sponge surface due to a large number of amino groups, and a high specific surface area (952.5 m2g-1), the resulting CNPFs-Col sponge with an optimized composition can activate the intrinsic pathway of the coagulation cascade, induce the adhesion of hemocytes and platelets, promote blood coagulation, and achieve bleeding control in vitro and in vivo. In addition, the CNPFs-Col sponge can be completely biodegraded within 3 weeks, which is suitable for postoperative treatment and prevention of peritoneal adhesions.

Chitosan with a deacetylation degree of 82.5%, bovine tendon, pepsin from the mucous membrane of the porcine stomach, lysozyme, collagenase type I and dialysis bags (membrane molecular weight cutoff of 3, 8 and 12-16 kDa), glacial acetic acid, CCK-8 kit, citric acid, sodium citrate, d-glucose, calcium chloride, sodium carbonate were used to create the hemostatic sponge.

CPNFs were successfully synthesized using an improved one-pot preparation method. Upon addition of calcium tripyrophosphate _{, tiny Ca2P2O7 crystals were} formed, forming a well-defined matrix. Chitosan molecules were then adsorbed onto the anisotropic nanocrystals, resulting in the formation of hybrid nanoparticles. The average diameter of CPNFs was calculated to be 322 nm.

In summary, a collagen sponge reinforced with chitosan/calcium pyrophosphate nanoparticles was developed for rapid bleeding control. The resulting CPNFs-Col sponge with optimized composition had the ability of rapid water absorption, a positive surface area rich in amino groups, and a high specific surface area. The CNPFs-Col sponge could activate the intrinsic pathway of the blood coagulation cascade, induce the adhesion of hemocytes and platelets, promote blood coagulation, and achieve rapid bleeding control.

Undoubtedly, the prototype has proven its effectiveness, however, it is not without a number of shortcomings. Collagen does not have its own adhesive properties, and chitosan in the prototype is used as nanoparticles for reinforcing the sponge, which also does not affect the increase in adhesiveness. As stated above, all collagen sponges have poor adhesion to the wound surface and can lose contact with the wound, which can potentially lead to relapse of bleeding. To fix the prototype sponge, it is necessary to use additional materials: gauze or other circular bandage, plaster, disposable wound bandage with an adhesive surface, etc.

It should also be noted that calcium phosphates were used in the creation of the hemostatic sponge, which can be potentially toxic, especially when using large quantities of the hemostatic sponge, for example in the case of extensive damage (Ksenzenko V.I., Stasinevich D.S. Calcium phosphates // Chemical encyclopedia: in 5 volumes / Ed. - in-chief I.L. Knunyants. - M .: Soviet encyclopedia, 1990. - V. 2: Daffa - Medi. - P. 299. - 671 p.). Thus, the prototype is also not intended to stop bleeding from internal organs and cannot be used in the case of massive bleeding.

The current task today is to find the most effective solution to the problem of stopping bleeding both in external bleeding and in case of damage to parenchymal organs.

The technical result of the present invention consists in creating an effective hemostatic agent with increased adhesion (including to a wet wound surface) that does not require additional materials for its fixation.

This result is achieved by the fact that a hemostatic agent has been created containing chitosan derivatives and hydrolyzed collagen, according to the invention, its salt and additionally tannin and arginine, or oligoarginine, or polyarginine, or protamine sulfate are used as chitosan derivatives in the following ratio of components, mass %:

chitosan salt 2-4 tannin 1 arginine 1 or oligoarginine 1 or polyarginine 1 or protamine sulfate 1 hydrolyzed collagen 20 water the rest

The hemostatic agent according to paragraph 1 is made in the form of a film, for the production of which chitosan salts, arginine, or oligoarginine, or polyarginine, or protamine sulfate, tannin and hydrolyzed collagen are dissolved in water and dried at room temperature on a flat surface for 48 hours in a fume hood.

The collagen molecule consists of three polypeptide chains that form a polyproline helix. Collagen is a protein of the extracellular matrix in the body, its function is to provide mechanical strength to the tissue. Thanks to it, tissues resist mechanical deformation. Collagen has low antigenicity (Gan K, Li J, Wu W, Xia C, Zhang T, Bi M, Zhou K, Li J, Xu D. Effect of Absorbable Collagen Sponge and Steroid Injection for 1- or 2-Segment Anterior Cervical Discectomy and Fusion: A Retrospective Comparison Study. World Neurosurg. 2022 Aug;164:e574-e581. doi: 10.1016/j.wneu.2022.05.009. Epub 2022 May 10. PMID: 35552033). Among the positive properties of collagen, it is also worth noting that the degradation products of collagen lead to the formation of amino acids that have thrombogenic potential and activate the coagulation cascade.

The use of chitosan salt in the present invention makes the film biologically inert and allows it to be left on the wound after the completion of surgery on parenchymatous organs and vessels. Organic and monobasic inorganic acids, such as acetate, lactate, succinate, malate, citrate and the like, can be used to create chitosan salt. When dried, chitosan forms a film that has adhesive properties, but which, when there is excess liquid, swells and becomes gelatinous, thereby increasing the adhesive properties of the proposed invention.

Additionally, the product contains arginine, an amino acid containing a positively charged guanidine group, which enhances the adhesive and hemostatic properties of chitosan. The Chitosan/Arginine film demonstrated suppression of the growth of E. Coli and S. Aureus colonies. Moreover, the chitosan and arginine solution did not exhibit cytotoxicity and promoted cell proliferation (Song J, Feng H, Wu M, Chen L, Xia W, Zhang W. Preparation and characterization of arginine-modified chitosan/hydroxypropyl methylcellose antibacterial film. Int J Biol Macromol. 2020 Feb 15;145:750-758. doi: 10.1016/j.ijbiomac.2019.12.141. Epub 2019 Dec 17. PMID: 31857168).

The adhesive properties of the film and resistance to soaking in a humid environment are enhanced by adding tannin, a plant polyphenolic tanning agent, to the wound contact layer. The good adhesiveness of tannin is due to its chemical reaction with nucleophilic groups such as _-NH2 or -SH on the tissue surface. The addition of tannin to the solution provided significant adhesion force to wet tissue and good cytocompatibility (Guo J, Sun W, Kim JP, Lu X, Li Q, Lin M, Mrowczynski O, Rizk EB, Cheng J, Qian G, Yang J. Development of tannin-inspired antimicrobial bioadhesives. Acta Biomater. 2018 May; 72:35-44. doi: 10.1016/j.actbio.2018.03.008. Epub 2018 Mar 17. PMID: 29555464; PMCID: PMC6328059). Tannin-based biological adhesives have antioxidant, anti-inflammatory and antibacterial properties. Tannin can scavenge free radicals and also promote proliferation of mammalian cells. The anti-inflammatory properties of tannin are due to its ability to reduce the expression of interleukin 1B and tumor necrosis factor alpha (Wu K, Fu M, Zhao Y, Gerhard E, Li Y, Yang J, Guo J. Anti-oxidant anti-inflammatory and antibacterial tannin-crosslinked citrate-based mussel-inspired bioadhesives facilitate scarless wound healing. Bioact Mater. 2022 May 21;20:93-110. doi: 10.1016/j.bioactmat.2022.05.017. PMID: 35633874; PMCID: PMC9131258). Tannin has no toxicity, which was studied when using dressings containing tannin in patients with extensive burns (Chokotho L, van Hasselt E. The use of tannins in the local treatment of burn wounds - a pilot study. Malawi Med J. 2005 Jun;17(1):19-20. doi: 10.4314/mmj.v17i1.10866. PMID: 27528993; PMCID: PMC3346037).

There is a variant of the invention where protamine sulfate is used as an additive to chitosan and tannin in the active layer - a protein in which 67% of the amino acid composition is represented by arginine, which makes it a highly alkaline polycationic substance with a molecular weight of 4.5 thousand Da. Multiple positively charged chains of protamine provide a strong electrostatic interaction with negatively charged groups on cell membranes.

Oligoarginine is a peptide containing an arginine region and possessing chemical properties similar to arginine (KASHEVEROV, I. E., KRYUKOVA, E. V., IVANOV, I. A., LEBEDEV, D. S., SPIROVA, E. N., EGOROVA, N. S., & TSETLIN, V. I. (2019). Oligo-arginine peptides as a new class of ligands of nicotinic acetylcholine receptors. Bulletin of the Far Eastern Branch of the Russian Academy of Sciences, (6s), 44).

Polyarginine is a positively charged synthetic polyamino acid (Fuchs SM, Raines RT. Polyarginine as a multifunctional fusion tag. Protein Sci. 2005 Jun;14(6):1538-44).

Due to its biological integrity and biodegradability, the preparation can be used for wounds of parenchymatous organs such as the liver, kidneys and spleen as a means of temporary or final hemostasis. After surgery, the preparation can be left in the abdominal cavity on the wound.

When making the film, a solution of collagen, chitosan salt, tannin and arginine is dried on a flat surface for 48 hours at room temperature in a fume hood. After drying, the plate is ready for use.

Drying in a fume hood to obtain a dry plate is chosen because in a wet state in the form of a hydrogel the agent does not have sufficient adhesion: in this case there is no tight contact of positively charged groups with the cell surfaces. The use of a dry film leads to the fact that at the point of its contact with biological tissue, local dehydration of the tissue surface occurs and tight contact is formed between the functional groups of the film components and the cell membrane.

The plate is applied to the bleeding surface of a parenchymatous organ, vessel, external or internal wound. Then the film is pressed tightly to the wound surface for 1 minute. After that, the film remains on the surface of the injury for the required period. At the moment of contact with the wound surface, its surface is rapidly dehydrated and electrostatic bonds are formed between the positively charged amino groups of chitosan and guanine groups of arginine with negatively charged molecules of cell membranes, as well as between the trihydroxybenzoic residues of tannins with charged groups of proteins of the wound surface.

The essence is explained by examples.

Example #1

1. A 2 wt.% aqueous solution of chitosan acetate is obtained (2 g of chitosan and 2 g of glacial acetic acid are added to 95 g of deionized water). The solution is stirred for 10 hours until the chitosan is completely dissolved.

2. Prepare an aqueous solution of hydrolyzed collagen. To do this, 20 g of hydrolyzed collagen obtained by enzymatic hydrolysis is dissolved in 76 g of water, then the solution is stirred for an hour until the collagen is completely dissolved.

3. The resulting chitosan acetate in the amount of 2 g is added to an aqueous solution of collagen, then 1 g of arginine is added to the solution, stirred on a magnetic stirrer until completely dissolved for about 2 hours. The resulting solution is heated to 60 ° C, after which 1 g of tannin is added with constant stirring on a magnetic stirrer, stirring at a temperature of 60 ° C until the latter is completely dissolved for 2 hours. The solution is dried at room temperature on a flat surface for 48 hours. After drying, the film is ready for use. Composition and content of components, mass %:

Hydrolyzed collagen 20 Chitosan acetate 2 Arginine 1 Tannin 1 Water the rest

Example #2

All stages are similar to example #1. The difference is that 2 g of hydrochloric acid is used to form chitosan salt. Composition and content of components in the wound contact layer, wt.%:

Hydrolyzed collagen 20 Chitosan chloride 2 Arginine 1 Tannin 1 Water the rest

Example #3

All stages are similar to example #1. The difference is that 4 g of citric acid is used to form chitosan salt. Composition and content of components in the wound contact layer, wt.%:

Hydrolyzed collagen 20 Chitosan citrate 2 Arginine 1 Tannin 1 Water the rest

Example #4

All stages are similar to example #1. The difference is that 4 g of lactic acid is used to form chitosan salt. Composition and content of components in the wound contact layer, wt.%:

Hydrolyzed collagen 20 Chitosan lactate 2 Arginine 1 Tannin 1 Water the rest

Example #5

All stages are similar to example #1. The difference is that 2 g of ready-made chitosan succinate dissolved in water is used. Composition and content of components in the wound contact layer, wt.%:

Hydrolyzed collagen 20 Chitosan succinate 2 Arginine 1 Tannin 1 Water the rest

Example #6

All stages are similar to example #1. The difference is that 1 g of protamine sulfate is used instead of arginine. Composition and content of components in the wound contact layer, wt.%:

Hydrolyzed collagen 20 Chitosan acetate 2 Protamine sulfate 1 Tannin 1 Water the rest

Example #7

All stages are similar to example #1. The difference is that 1 g of polyarginine is used instead of arginine. Composition and content of components in the wound contact layer, wt.%:

Hydrolyzed collagen 20 Chitosan acetate 2 Polyarginine 1 Tannin 1 Water the rest

Example #8

All stages are similar to example #1. The difference is that 1 g of oligoarginine is used instead of arginine. Composition and content of components in the wound contact layer, wt.%:

Hydrolyzed collagen 20 Chitosan acetate 2 Oligoarginine 1 Tannin 1 Water the rest

Thus, the combination of the above-described components in the specified quantities provides a pronounced hemostatic effect and increased adhesion of the agent with biological inertness and the ability to biodegrade, which allows it to be used without additional fixing materials, and also ensures the versatility of its application: the invention can be used for massive bleeding both with external wounds and with damage to parenchymatous organs and vessels.

The developed film, due to its excellent adhesive properties, allows it to be used without additional fixation.

The addition of arginine and tannin to the preparation, in addition to enhancing its adhesive properties, improves its beneficial properties without increasing the number of side effects.

Claims (3)

1. A hemostatic agent containing chitosan derivatives and hydrolyzed collagen, characterized in that the chitosan derivatives used are its salt and additionally tannin and arginine, or oligoarginine, or polyarginine, or protamine sulfate, in the following ratio of components, mass %: chitosan salt 2-4 tannin 1 arginine 1 or oligoarginine 1 or polyarginine 1 or protamine sulfate 1 hydrolyzed collagen 20 water the rest 2. A hemostatic agent according to paragraph 1, characterized in that it is made in the form of a film.