WO2021194461A1

WO2021194461A1 - The method of production of a haemostatic agent and a haemostatic agent for major bleeding control

Info

Publication number: WO2021194461A1
Application number: PCT/UA2021/000031
Authority: WO
Inventors: Serhiy Vassilliovytch KOMISARENKO; Volodymyr Hryhorovych NIKOLAEV; Tetyana Mykolaivna PLATONOVA; Viktor Yevhenovych DOSENKO; Larysa Oleksiivna SAKHNO; Elizaveta Oleksandrivna SNEZHKOVA; Tamara Martynivna CHERNYSHENKO; Daria Serhiivna KOROLOVA; Volodymyr Oleksandrovych CHERNYSHENKO; Olha Volodymyrivna HORNYTSKA; Valentyna Hryhorivna KOROTYCH
Original assignee: Palladin Instytute Of Biochemistry Of The National Academy Of Sciences Of Ukraine
Priority date: 2020-06-22
Filing date: 2021-03-29
Publication date: 2021-09-30

Abstract

The invention belongs to medicine, including, but not limited to, surgery and haematology, and relates to the method of production of a haemostatic agent in which a dressing material is prepared; the solution of an active haemostatic component is prepared that includes ekamulin - an enzymatic activator of the blood coagulation system, isolated from the venom of Echis multisquamatus; the solution of an active haemostatic component is applied to the prepared dressing material, and the active haemostatic component is non-covalently immobilized with a further freezing and lyophilizing to obtain a haemostatic agent; and the mentioned haemostatic agent is sterilized by radiation, as well as an effective haemostatic agent prepared according to the method to control major bleeding, in case of haemophilia as well, comprising a dressing material and an active haemostatic component.

Description

THE METHOD OF PRODUCTION OF A HAEMOSTATIC AGENT AND A HAEMOSTATIC AGENT FOR MAJOR BLEEDING CONTROL

Branch of the invention

The invention belongs to medicine, including, but not limited to, surgery and haematology, and relates to the method of production of a haemostatic agent and an effective haemostatic agent for rapid control of major bleeding during surgical treatment, primary medical care in case of injuries and external bleeding due to haemophilia.

Major bleeding is one of the main causes of death due to injuries and wounds during natural and man-made disasters, as well as in case of haemophilia, so the production of new, reliable and safest agents with a significant haemostatic effect to ensure timely control of major bleeding is one of the priorities of modem medicine.

Haemostatic agents have a number of requirements that they must meet, including the ensuring of complete bleeding control in the shortest possible time, the presence of a sufficiently high adhesiveness, the ability to fit tightly to the wound surface, the prevention of bleeding recurrence, the absence of irritant effect on tissues at the place of application, the absence of toxicity, the absence of the effect on haemostasis function in the general circulation, the ease of use, such as the ability to evenly and simultaneously cover the wound surface and the ease of removal from the wound after application, the maintaining of the agent’s activity for a long period of time and its sterility.

Background of the invention

There are several groups of haemostatic agents: based on collagen and/or gelatine (an absorbable sponge “Cutanplast”, gelatine sponges “Spongostan” and “Gelfoam”, collagen haemostatic sponge [1-4]), based on kaolin, cephalin (QuikClot® [5 -8]) and fibrin-containing haemostatic agents (“Fibrinostat” [9]), as well as based on chitosan (“CHITOGAUZE XR PRO” [10, 11]) and batroxobin-containing hydrogel SB50, created from synthetic nanofibres and filled with batroxobin - a component of the venom of two species of South American rattlesnake [12].

The main disadvantage of these haemostatic agents is that they initiate blood coagulation non-specifically as a result of a chemical or physical reaction, which leads to their short-term action and makes them ineffective for major bleeding.

From the background of the invention [13, 14], the haemostatic agents based on textile dressing materials with an active haemostatic component, such as haemostatic dressings with salts of alginic acid and Furagin (Coletex-Hem), with aminocaproic acid and Feracryl (AktivtexAKF and Aktivtex-FHem), with chitosan (Revul, HemCon dressing), with iron gluconate (Hemotex), with kaolin (QuikClot Combat Gauze (QCG) and QuikClot Combat Gauze XL (QCX), with mollusc chitin (Celox) and fibrin sealing dressings, are known.

Haemostatic agents based on non-woven materials are more effective; in particular, they fit tightly and provide adequate blood sorption, non-invasiveness and prevention of infection in the wound. However, active haemostatic components used in known haemostatic agents based on non-woven materials also initiate blood coagulation non-specifically as a result of a chemical or physical reaction, so do not solve the problem of effective and long-term control of major bleedings.

From the background of the invention, the enzymatic activator of the blood coagulation system - ekamulin, isolated from the venom of Echis multisquamatus, which directly activates prothrombin [15], is known. Ekamulin specifically initiates blood coagulation by providing direct activation of a key proenzyme of the blood coagulation system - prothrombin, which leads to the formation of thrombin with a subsequent formation of autogenous covalently stabilized fibrin and thus the ensuring of the control of bleeding caused by surgical treatment or injury. However, ekamulin due to its protein nature is sensitive to the effects of negative environmental factors and loses biological activity during long-term storage [15], which complicates its use.

The closest analogue of the claimed invention is a haemostatic agent comprising a dressing material selected from activated fibre carbon-base material for medical purposes, on which the enzymatic activator of the blood coagulation system isolated from the venom of Echis multisquamatus is non-covalently immobilized. Also the methods of preparing such a haemostatic agent were described, which involved the application of 2 or 8 pg of the enzymatic activator of the blood coagulation system isolated from the venom of Echis multisquamatus and 2x1 O ⁴ M of CaCl₂ to 1 cm² of the activated fibre carbon-base material of the brand “Dnepr”-MP [16]. However, in a further study of this haemostatic agent, it was found that for a short time it loses its initial haemostatic characteristics and has a limited enough shelf life. In addition, the agent described in this source did not belong to sterile drugs, which is very important when working with large open wounds and direct contact with a patient’s blood.

Thus, the development of effective haemostatic agents that combine the specificity of the action of the enzymatic activator of the blood coagulation system and long-term storage without loss of activity remains an urgent task.

Brief description of the invention

The claimed invention is based on the task of developing a method of production of an effective haemostatic agent based on ekamulin, an enzymatic activator, as a powerful haemostatic agent to control major bleeding with a long shelf life.

The set task is solved in accordance with the method of production of a haemostatic agent to control major bleeding, in case of haemophilia as well, comprising a dressing material and an active haemostatic component, in which a dressing material is prepared, the solution of an active haemostatic component - ekamulin is prepared, the solution of an active haemostatic component is applied to the prepared dressing material, and the active haemostatic component is non- covalently immobilized with a further freezing and lyophilizing to obtain a haemostatic agent; and the haemostatic agent is sterilized by radiation.

In a separate embodiment of the claimed method, an activated fibre carbon- base material for medical purposes is used as a dressing material.

In a separate embodiment of the claimed method, the preparation of the dressing material includes rinsing with distilled water, giving the required size and shape, and heat treatment.

In a separate embodiment of the claimed method, the heat treatment is performed in a drying oven at a temperature between 80 and 120°C, preferably at a temperature of 90°C, for 1-2 hours.

In a separate embodiment of the claimed method, the preparation of the solution of an active haemostatic component involves dissolving the lyophilisate of ekamulin in 0.9% aqueous sodium chloride solution containing the excipient calcium chloride in the amount of 10⁴- 10³ M, to a final concentration of ekamulin of 0.7 - 1.0 pg/ml.

In a separate embodiment of the claimed method, the preparation of the solution of an active haemostatic component additionally involves sterilization under aseptic conditions by passing through a sterilizing filter, preferably through a sterilizing filter with a pore size of 0.2 mih.

In a separate embodiment of the claimed method, the solution of an active haemostatic component is applied in the amount of 8 ± 0^'5 pg of ekamulin to 1 cm of prepared dressing material.

In a separate embodiment of the claimed method, after immobilization of an active haemostatic component, freezing is performed at a temperature of -20 ± 2°C for 12-15 hours.

In a separate embodiment of the claimed method, after freezing, lyophilisation is performed at a condenser temperature of -83°C and a pressure of 0.05 millibars for 12 hours. In another preferred embodiment, the claimed method additionally includes packaging of the haemostatic agent in a sealed package before performing sterilization by radiation.

In an additional embodiment of the claimed method, the sterilization by radiation is performed with a radiation dose of up to 25 kGy.

In a separate embodiment of the claimed method, the obtained haemostatic agent is an application haemostatic agent.

The claimed invention also provides a haemostatic agent to control major bleeding, in case of haemophilia as well, obtained in accordance with the method described in this application, according to which a dressing material is prepared; the solution of an active haemostatic component - ekamulin is prepared; the solution of an active haemostatic component is applied to the prepared dressing material, and the active haemostatic component is non-covalently immobilized with a further freezing and lyophilizing to obtain a haemostatic agent; and the mentioned haemostatic agent is sterilized by radiation

In a separate embodiment of the claimed method, the preparation of the solution of an active haemostatic component involves dissolving the lyophilisate of ekamulin in 0.9% aqueous sodium chloride solution containing the excipient calcium chloride in the amount of lO^-lO ³ M, to a final concentration of ekamulin of 0.7 - 1.0 pg/ml.

In a separate embodiment of the claimed method, the preparation of the solution of an active haemostatic component additionally involves sterilization under aseptic conditions by passing through a sterilizing filter, preferably through a sterilizing filter with a pore size of 0.2 pm.

In a separate embodiment of the claimed method, the solution of an active haemostatic component is applied in the amount of 8 ± 0.5 pg of ekamulin to 1 cm of prepared dressing material.

In a separate embodiment of the claimed method, after freezing, lyophilisation is performed at a condenser temperature of -83°C and a pressure of 0.05 millibars for 12 hours.

In another preferred embodiment, the claimed method additionally includes packaging of the haemostatic agent in a sealed package before performing sterilization by radiation.

In a separate embodiment of the claimed method, the haemostatic combined agent is an application haemostatic combined agent.

The technical result of the claimed invention is to create a method of obtaining a haemostatic agent, which achieves the preservation of high stable haemostatic activity of ekamulin, the enzymatic activator, for a long period of storage of the agent.

This technical result is achieved by producing a haemostatic agent based on ekamulin, the enzymatic activator, using the claimed method. Lyophilized enzymatic activator, non-covalently immobilized on the dressing material, is stable and retains activity for a long shelf life, which provides optimal characteristics of the final product.

Obtained in accordance with the claimed method, the haemostatic agent allows controlling minor and intense blood loss, protecting the bleeding site from infection through sterility, improving blood microcirculation, which reduces oedema and leads to rapid wound healing, and can be at the site of bleeding for the time required, maintaining sufficient haemostatic activity.

Brief description of the drawings of the invention

Figure 1 shows a possible variant of the technological scheme, according to the method of production of a haemostatic agent.

Detailed description of the invention

The method of production of the claimed effective haemostatic agent involves the provision and preparation of a dressing material and an enzymatic activator of the blood coagulation system - ekamulin.

The dressing material according to the invention is a material suitable for the use in operations and dressings to dry an operating field and a wound, for wound tamponade with the purpose of bleeding control and drainage, to apply dressings, as well as to protect a wound from secondary infection and damage. The dressing material can be made of natural and synthetic materials, in particular of wood, cotton, paper and viscose yam, etc. The main requirements for the dressing material are sufficient strength, elasticity, safety for an organism, high hygroscopy or absorption capacity, capillarity, neutrality, as well as the ability to withstand sterilization. The dressing material may be woven or nonwoven, and made in different shapes and sizes, depending on the use, such as a bandage, napkin, ball, tampon, etc.

The dressing materials, particularly preferred for the use according to the invention, are activated fibre carbon-base materials, such as of the brand AUVM “Dnepr”-MN TC U 88.023.026 or the brand AUT-M (3) TC RB 00204056-076, with a sorption surface not less than 1500 cm /g [17], which perform two functions, namely: a matrix for strong non-covalent immobilization of the enzymatic activator of the blood coagulation system and an adsorption component, which provides effective absorption from the wound surface of toxic substances of different molecular weight, including products of proteolysis and thermal denaturation of proteins, biogenic amines and inflammatory mediators, bacterial toxins, and, as a consequence, improvement of local haemostasis, relief of traumatic oedema, reduction of local and general inflammatory response intensity, prevention of complications, acceleration of regenerative processes and reduction of the term of wound healing [18 - 20].

The preparation of the dressing material for non-covalent immobilization of ekamulin involves a number of treatments aimed at improving the material quality and sterility. Such treatments can be, in particular, rinsing with liquids, heat treatment from about 80°C to 120°C, in particular under conditions of high pressure, providing the desired moisture content, giving the desired shape and size. It is understood that the shape and size of the dressing material, and accordingly the finished haemostatic agent, is primarily due to its application; in particular, when used to control major bleeding, the dressing material is made in the form of a bandage, which has a form of a z-shaped accordion and has a total size: width - (5-10) cm, length - (50-100) cm; for other uses, it may have the following shapes and sizes: for dressing - (5 x 5), (5 x 10) and (10 x 10) cm; for turunda - (0.2 - 1.5) x (1 - 10) cm.

The preferred treatment of the dressing material according to the invention is rinsing with distilled water to remove mechanical contaminants, cutting the dressing material according to the specified sizes and drying in an oven (e.g., a drying sterilization cabinet USO-80g UHL 42, “Biomed”, Ukraine ) at a temperature of about 90°C for about 2 hours.

Ekamulin is isolated from crude or lyophilized venom of Echis multisquamatus. For the purposes of this invention a crystalline preparation of lyophilized venom of Echis multisquamatus was used, which is produced by the laboratory of experimental herpetology, the JSC “Trypillia Biochemical Plant” (Ukraine).

To obtain ekamulin, a crystalline preparation of the venom is dissolved in 0.025 M of tris-HCl buffer, pH 8.9, to a concentration of 50 mg/ml and after centrifugation is applied to Q-Sepharose, 1x2 cm (Sigma, USA), equilibrated with 0.025 M of tris-HCl buffer, pH 8.9. Sorbed proteins are eluted in a step gradient of 0.125; 0.2; 0.35; 0.5; 1M of NaCl in a buffer at a rate of 1 ml/min. The venom fraction having clotting activity is obtained and lyophilized at a condenser temperature of -83°C and a pressure of 0.05 millibar for 12 hours, for example using Telstar equipment (LyoQuest, Spain). The obtained lyophilized ekamulin is a white powdery substance that is readily soluble in aqueous solutions. Ekamulin is a two-subunit protein with a molecular subunit weight of 57 and 22 kDa, which is able to specifically hydrolyze a chromogenic substrate of kallikrein D-Pro - Phe - Arg ~ pNA (Chromogenix, Sweden), does not hydrolyze fibrinogen and does not activate platelets.

The preparation of the enzymatic activator solution involves dissolving in a suitable vessel a given amount of lyophilized substance of ekamulin in the required amount of physiological solution (0.9% aqueous sodium chloride solution (“Damytsia”, Ukraine), additionally containing 10^~4-10³ M of calcium chloride (10% calcium chloride solution, Brovapharma, Ukraine), to the ekamulin concentration of 0.7 pg/ml, followed by sterilization by passing through a sterilizing filter with a pore size of 0.2 pm (MEDICARE, China) under aseptic conditions. The addition of calcium chloride is intended to provide stabilization and covalent insertion of a blood clot. It is clear that the enzymatic activator solution may, if necessary, contain one or more additional components, in particular selected from buffer solutions, stabilizers, preservatives, polymers, additional active components, etc., which may be useful during implementation of the method of obtaining a haemostatic agent, or the presence of which may be useful in the agent itself.

The application of the enzymatic activator involves its non-covalent immobilization on the surface of the prepared dressing material, which occurs due to the sorption force of the dressing material and is carried out by the methods well known in this branch of technology, which ensure uniform distribution of the sterile enzymatic activator solution on the plane of the prepared dressing material. Uniform distribution can be performed both on the entire surface of the prepared dressing material, and only on the part of its surface intended for contact with a wound. Such methods may include, in particular, immersion of the prepared dressing material in the sterile enzymatic activator solution, drip application of the sterile enzymatic activator solution on the surface of the prepared dressing material using a repeater Repeater M4 (Eppendorf, USA), and others. When applied, the amount of 8 ± 0.5 pg of the enzymatic activator on 1 cm² of the prepared dressing material is provided.

After the application procedure, the dressing material with enzymatic activator is subjected to pre-freezing in a freezing chamber, for example in a freezing chamber HR1-700T-3 (Esco, USA), at a temperature of -20 ± 2°C for 12- 15 hours, followed by lyophilisation at a condenser temperature of -83°C and a pressure of 0.05 millibars for 12 hours, for example using Telstar equipment (LyoQuest, Spain).

After the lyophilisation, the haemostatic agent is packaged in an individual sealed package and a consumer packaging. The individual sealed package must ensure the sealed storage of the product during its shelf life and can be made of any suitable material, such as glass or polymeric film material, such as polyethylene film (Hendi, the Netherlands). In case of the use of polymeric film material, the package sealing is ensured by welding with equipment known in this field, such as electrical contact welding device “Molnia - 3”, TC 16-239.206-79, USSR. The size of the individual package may vary depending on the size of the product according to the present invention, for example, may be 100x150 mm, but is not limited thereto. Individual packages are packed in consumer packaging, for example, in bags made of paper, composite materials or bags made of plastic film or other materials approved for the use by the Ministry of Healthcare of Ukraine. The consumer packaging may have the required printed marking and/or label.

The marking on the packaging and/or label shall indicate the necessary information, in particular the name of the manufacturing facility, its address and trademark, legal address; product name; linear dimensions, cm x cm; date of production and batch number; designation of technical specifications TC; number and date of the state registration certificate; signs indicating: radiation sterilization of the product, shelf life and storage conditions; mark of conformity according to the DSTU 2296 (at certification); brief instructions for use. The list of information intended to be placed on the label may be different and depends on the requirements of the law.

The sterilization of the haemostatic agent is carried out after its packaging in an individual package or in consumer packaging by radiation method in accordance with the DSTU EN ISO 11137-2:2015 (“Sterilization of health care products — Radiation — Part 2: Establishing the sterilization dose ”); DSTU EN ISO 9001-2015; DSTU EN ISO 13485:2015. The sterilization of the haemostatic agent is carried out using specialized equipment known in this field for irradiation, for example, an electron accelerator (Russia) using a radiation dose of up to 25 kGy. The shelf life of the haemostatic agent after sterilization is at least 2-3 years.

The product sterility control is carried out by the methods well known in this field, for example in accordance with an industry standard OST 42-21-2-85 “Sterilization and disinfection of medical devices. Methods, means and modes”, under aseptic conditions that exclude the possibility of secondary contamination of products by microorganisms. The sterility control is carried out by direct inoculation (immersion of the product without packaging, rinsing from the product without packaging) of standard nutrient media for sterility control, such as thioglycollate medium (TITAN BIOTECH LTD, India), Saburo medium (LLC “Farmaktyv”, Ukraine). The composition of the media and the method of their preparation are standard and known to specialists in this field, in particular contained in the Annex No. 1 to the order of the Ministry of Healthcare of Ukraine No. 720 as of 31 July 1978 “On improving medical care for patients with purulent surgical diseases and strengthening measures to fight against healthcare- associated infections” (thioglycollate medium with a dry nutrient medium is prepared in the manner indicated on the label). During the product sterility control, a simultaneous inoculation of both specified nutrient media is obligatory. The inoculations of thioglycollate medium are kept in a thermostat at a temperature of 32°C, the inoculations of Saburo medium - at a temperature of 20- 22°C for 7-14 days. In the absence of the growth of microorganisms in all samples, a conclusion about the sterility of the products is made.

The sterility of the finished product was ensured at several stages of its preparation, starting from the preparation of the dressing material and the enzymatic activator solution, and at the final stage of sterilization of the finished product in the consumer packaging. As a result of the method implementation according to the invention, the haemostatic agent was obtained in which the stability and high activity of the enzymatic activator are maintained for a long shelf life due to lyophilisation.

In the context of this invention, the term “bleeding” is defined as the outflow of blood from blood vessels when damaging their integrity. Bleeding can be both insignificant and intense. Bleeding is also characterized by its nature; signs of arterial bleeding: rapid and significant bleeding (blood is “gushing forth”, pulsating, bright red), which leads to significant blood loss in a short time; signs of venous bleeding from a wound: dark red blood flows continuously from a wound; depending on the diameter of the damaged vein, bleeding can be from insignificant to intense.

The haemostatic agent according to this invention can be successfully used to control both insignificant and intense bleeding, in particular major bleeding.

Examples of implementation of the invention

The following are the examples that confirm the possibility of carrying out the claimed invention to obtain the specified technical result. Example 1

Determination of activity of the enzymatic activator of the blood coagulation system in buffer medium and in physiological solution.

It is known that enzyme preparations, in particular the highly purified preparation of ekamulin, retain 100% of activity in buffer solution, and the change in pH of the solution can lead to a sharp decrease in enzymatic activity [13]. The use of buffer solutions may be unsuitable due to the need for additional special preparation and the introduction of additional salts. Therefore, the suitability of the use of standardized sterile physiological solution, which has a stable composition and is approved for the use in medicine, for the dissolution of lyophilized enzymatic activator was tested. This example compares the activity of the enzymatic activator in buffer solution of 0.05 M of tris-HCl (Sigma, USA) with 0.13 M of NaCl (Sigma, USA), pH 7.4, and in sterile physiological solution - 0.9% aqueous sodium chloride solution.

The tested solutions of the enzymatic activator of ekamulin were prepared by dissolving in vitro a portion of the lyophilized sample in these dissolution media - buffer solution and sterile physiological solution, to a concentration of ekamulin of 1 pg/ml.

The study of the amidolytic activity of ekamulin, which is determined by the ability of proteinase to hydrolyze amide bonds and indicates functional activity, was determined as follows: 10 mΐ of the enzymatic activator solution (at a concentration of 1 pg/ml) in 0.05 M of tris-HCl buffer, pH 7.4, with 0.13 M of NaCl or in sterile physiological solution (Damytsia, Ukraine) - 0.9% aqueous sodium chloride solution was incubated with 25 mΐ of 2 mm chromogenic substrate S2302 (Chromogenix, Sweden) for 5 min at 37°C in the well of the microplate (MICROLON (Greiner Bio-One, Germany)). The optical absorption was then determined using a microplate reader Multiscan EX (ThermoFisher Scientific, USA) at a wavelength of 405 nm; the results of the study are presented in the Table 1 below. The optical absorption at a wavelength of 405 nm corresponds to the concentration of «-nitroaniline, which is split out of the chromogenic substrate by the action of the enzyme.

It is shown that both in physiological solution and in buffer system, the enzymatic activator effectively hydrolyzes the chromogenic substrate S2302, as indicated by the extremely close values of optical absorption of the incubation medium for the same period of time.

Table 1

The amidolytic activity of the enzymatic activator of the blood coagulation system

The activity of the enzymatic activator in relation to blood coagulation was determined as follows: 100 mΐ of the enzymatic activator solution (at a concentration of 1 pg/ml) in 0.05 M of tris-HCl buffer, pH 7.4, with 0.13 M of NaCl and in sterile physiological solution (Damytsia, Ukraine) - 0.9% aqueous sodium chloride solution and 100 mΐ of human plasma were incubated at 37°C in a glass conical tube, recording the coagulation time; the results of the study are presented in the Table 2 below.

Table 2

The coagulation time under the action of the enzymatic activator of the blood coagulation system

It is shown that the enzymatic activator effectively performs its function of stimulating blood coagulation in both buffer solution and physiological solution, which allows the use of sterile physiological solution to dissolve the enzymatic activator in the claimed method without losing its biological activity. The use of certified sterile physiological solution (0.9% sodium chloride solution for infusions, Damytsia, Ukraine), which is approved for medical use, prevents contamination of the finished haemostatic agent through a solvent.

Example 2

Determination of the activity of the enzymatic activator of the blood coagulation system before and after sterilization by passing through a sterilizing filter.

To achieve the desired level of sterility of the finished haemostatic agent, it is highly desirable that the enzymatic activator be sterile when applied to the prepared dressing material. In addition, residues of microorganisms or fragments thereof that may enter the product together with the enzymatic solution may be allergenic even in case of further sterilization and the absence of viable microorganisms. Therefore, the suitability of the sterilizing filter for sterilization of the enzymatic activator solution was checked. This example compares the activity of the enzymatic activator before and after sterilization of the solution by passing through a sterilizing filter.

The enzymatic activator solution of ekamulin was prepared by dissolving in vitro a portion of the lyophilized drug in sterile physiological solution - 0.9% aqueous sodium chloride solution to a concentration of ekamulin of 0.7 mg/ml.

The resulting solution was passed through a sterilizing filter with a pore size of 0.2 pm (MEDICARE, China). The ami do lytic and clotting activities of the enzymatic activator were determined similarly to the method described above in the Example 1, using the solution of ekamulin before and after the filtration through a sterilizing filter; the results of the activity study are shown in the Table 3 below. Table 3

The activity of the enzymatic activator of the blood coagulation system before and after filtration through a sterilizing filter

The loss of enzymatic activator activity after passing through a sterilizing filter was found to be insignificant, at the level of 6%, but this did not lead to any appreciable change in blood coagulation time, as might be expected given the protein nature of the activator. The results of this study prove that it is possible to apply sterilization of the enzymatic activator by passing through a sterilizing filter without noticeable loss of its biological activity.

Example 3

Determination and comparison of haemostatic ability of the haemostatic agent subjected to lyophilisation and a product made ex tempore

The obtaining ex tempore of the haemostatic agent is carried out as follows:

The enzymatic activator solution of ekamulin is prepared by dissolving in vitro a portion of the lyophilized preparation of ekamulin in physiological solution (0.9% aqueous sodium chloride solution) with the addition of an excipient in the amount of 10⁴-10³ M of calcium chloride (10% calcium chloride solution, Brovapharma, Ukraine), to a concentration of 0.7 pg/ml. The obtained enzymatic activator solution is applied by drop infusion using a repeater Repeater M4 (Eppendorf, USA) to the dressing material - activated fibre carbon-base material for medical purposes (AUVM “Dnepr-M”, Ukraine), ex tempore at the rate of 8 ± 0.5 gg of the enzymatic activator per 1 cm of the dressing material.

The obtaining of the haemostatic agent involving lyophilisation is carried out similarly to the above-described method of obtaining ex tempore haemostatic agent, which is supplemented by the stage of its lyophilisation, which involves pre-freezing in a freezer HR1-700T-3 (Esco, USA) at -20 ± 2°C for 12-15 hours, followed by lyophilisation on Telstar equipment (LyoQuest, Spain) at a condenser temperature of -83°C and a pressure of 0.05 millibars for 12 hours. This equipment has a built-in temperature control channel and according to the specified program, the temperature after the drying process gradually rises to ambient temperature (+21°C) and heats the product to the appropriate temperature.

The haemostatic ability of the above agents is compared on the classical model of arterial bleeding of rats, described in [22], with modifications. Experiments to simulate experimental bleeding from the carotid artery are performed for 16 Wistar rats; the average weight of animals was 250.2 ± 18.5 g (age of animals - 2 months ± 1 week). The animals divided into two groups are kept in a vivarium on a standard diet [22]. In 8 animals (the first group) bleeding from the carotid artery is simulated and a haemostatic agent made ex tempore is applied. In the second group (8 animals) for a similar injury a lyophilized haemostatic agent is used according to the invention. Both types of agents are pressed to the wound in 2-3 seconds after the occurrence of spontaneous bleeding from the left carotid artery and held for 3 minutes with a clamp. The clamp is then removed and a thorough collection of all blood flowing from the wound is performed, followed by weighing and calculation of blood loss, which is normalized by the weight of each animal and expressed as a percentage. The material used to control bleeding in animals is used according to the scheme of “blind” study.

The blood loss and mortality of animals, as well as the appearance of soluble fibrin in the bloodstream are evaluated one hour after the bleeding control. The soluble fibrin is considered a marker of intravascular coagulation [23].

The soluble fibrin is a specific marker of activation of the blood coagulation system. It is a soluble complex of fibrin oligomers with fibrinogen molecules circulating freely in the blood. An increase in the concentration of soluble fibrin indicates the appearance of thrombin in the bloodstream, the formation of monomeric fibrin, its complexes with fibrinogen and soluble oligomeric fibrin. The soluble fibrin is an early predictive indicator of the risk of intravascular thrombosis. The concentration of soluble fibrin in plasma characterizes the degree of activation of the blood coagulation system. In particular, its growth above 0.035 mg/ml indicates an extremely high risk of intravascular thrombosis [24]

The results of the study are presented in the Table 4 below.

Table 4

Comparison of a haemostatic agent subjected to lyophilisation and an agent made ex tempore in terms of blood loss, rat mortality and the level of soluble fibrin (SF) in the bloodstream

The results of the study indicate that the haemostatic agent produced ex tempore is unfit for use, has a higher blood loss rate, high rat mortality and an extremely high risk of intravascular thrombosis compared to the haemostatic agent subjected to lyophilisation. This difference is probably due to the effect of “washing off’ the enzymatic activator applied ex tempore into the bloodstream, as evidenced by the significant level of soluble fibrin in the blood plasma of all experimental animals one hour after the bleeding control. Thus, the enzymatic activator applied ex tempore, washing off acts not locally at the site of application to the wound, but in the general bloodstream, which impairs its haemostatic properties and, at the same time, threatens the development of thrombosis.

The traces of soluble fibrin in the blood plasma of rats treated with a lyophilized haemostatic agent are observed in only 29% of experimental animals and are a common consequence of surgical treatment that is not associated with the use of the agent itself [25].

The lyophilisation of the enzymatic activator, which is non-covalently immobilized on the dressing material, allows ensuring optimal characteristics of the final product, in particular maintaining and keeping the activity of the enzymatic activator and increasing the duration of its storage in the final product. In addition, in this case, the dressing material is likely to act as a stabilizer of the enzymatic activator.

Example 4

Comparison of the activity of the enzymatic activator non-covalently immobilized on the dressing material after sterilization by radiation with doses of 10, 15, 20 and 25 kGy

Sterilization by radiation is a generally accepted and standardized method of disinfecting medical dressings, but is not used to sterilize protein preparations, including enzyme preparations, due to the high risk of the loss of their activity.

It is known [21] that the biological action of radiation is based on the processes of ionization and excitation of molecules, radiation-chemical reactions that disrupt or alter the function and activity of enzymes, and high doses of radiation lead to inactivation of enzymes. Therefore, the study of the effect of radiation on the activity of the enzymatic activator non-covalently immobilized on a dressing material was performed.

The study used a haemostatic agent subjected to lyophilisation, obtained as described in the Example 3 above, which was subjected to additional sterilization by radiation (type of irradiation - accelerated electrons) with doses of 10, 15, 20 and 25 kGy and compared the activity of the enzymatic activator before and after irradiation.

The sterilization of the packaged product is performed on an electron accelerator ILU-6 (BINP SB RAS, Russia) in accordance with the DSTU EN ISO 11137-2:2015 “Sterilization of health care products — Radiation — Part 2: Establishing the sterilization dose”); DSTU EN ISO 9001-2015; DSTU EN ISO 13485:2015.

The activity of the enzymatic activator before irradiation is given in the Table 3. To assess the activity of the enzymatic activator after sterilization, the haemostatic agent is immersed in sterile physiological solution (0.9% aqueous sodium chloride solution) and the lyophilized enzymatic activator is dissolved to a final concentration of 1 pg/ml. To do this, a physiological solution is slowly added to a vial with a haemostatic agent containing a known amount of lyophilized enzymatic activator and is kept at room temperature until the lyophilized enzymatic activator is completely dissolved. The amidolytic and clotting activities of the enzymatic activator of the blood coagulation system are determined similarly to the method described in the Example 1. The results of the studies are presented in the Table 5 below.

Table 5

The activity of the enzymatic activator of the blood coagulation system non- covalently immobilized on the dressing material before and after irradiation

The effect of sterilization by radiation on the activity of the enzymatic activator non-covalently immobilized on a dressing material was studied for the first time. Unexpectedly, it was found that radiation doses of 10 and 15 kGy do not reduce the activity of the enzymatic activator non-covalently immobilized on the dressing material. Higher doses of radiation of 20 and 25 kGy do not significantly reduce the activity of the enzymatic activator. The results of the residual activity study are shown in the Table 6 below. Table 6

Residual activity of the enzymatic activator of the blood coagulation system non- covalently immobilized on the dressing material before and after irradiation

Therefore, the sterilization by radiation with a dose of up to 25 kGy can be used as the final stage of the method of production of a haemostatic agent according to this invention. However, radiation doses above 25 kGy can have a significant negative effect on the activity of the enzymatic activator of ekamulin.

It was not obvious that in the composition of the haemostatic agent obtained according to this invention, ekamulin is so stabilized that even at high doses of sterilizing radiation it retains structural and functional characteristics, in particular does not significantly lose the ability to activate prothrombin and initiate plasma coagulation.

Example 5

Comparative analysis of the stability of the lyophilized haemostatic agent according to this invention and of the ex tempore haemostatic agent obtained by the method according to the patent UA 117852 C2 for a long storage time.

The haemostatic agent according to this invention is prepared as described above and tested for stability in comparison with a control agent (ex tempore haemostatic agent obtained according to the patent UA 117852 C2). The term “stability” refers to the ability of the enzymatic activator of ekamulin to maintain its activity at the initial level for a long period of storage and not to lose it. An accelerated stability study is performed when storing samples of these agents at 8°C and 23°C for 1, 2 and 6 months and at a relative humidity of not more than 25%. The samples of the lyophilized haemostatic agent and control agent are placed in a polyethylene package and kept in the dark under the specified conditions for the specified time.

To analyse the stability of the enzymatic activator, all studied samples of the lyophilized haemostatic agent according to this invention and ex tempore haemostatic agent after storage are immersed in sterile physiological solution (0.9% aqueous sodium chloride solution) in separate vials; the enzymatic activator is dissolved to a final concentration of 0.8 mg/ml; the amidolytic and clotting activities of the enzymatic activator are determined as described in the Example 1.

The results of the study are shown in the Table 7 below; the amidolytic and clotting activities of the enzymatic activator are expressed as a percentage; the initial activity of the enzymatic activator is taken for 100%.

Table 7

Relative amidolytic (A) and clotting (B) activities of the enzymatic activator of the blood coagulation system during long-term storage

A

B

Thus, the enzymatic activator in the lyophilized haemostatic agent does not lose its initial activity, but retains it at the initial level during long-term storage at room temperature.

In view of the results given in the Table 7, it can be concluded that the lyophilized haemostatic agent obtained according to this invention has significant advantages over the ex tempore haemostatic agent according to the patent UA 117852 C2, namely it is more stable and retains its initial activity during longterm storage.

In addition, the appearance of thrombin in blood plasma under the action of the enzymatic activator is determined. The appearance of thrombin is fixed by the amidolytic activity of thrombin, which appears under the action of the activator, in the blood plasma as follows: 10 mΐ of plasma of healthy donors (Pooled Human Plasma, Innovative Research, USA) is incubated with 10 mΐ of the enzymatic activator solution (at the concentration of 1 pg/ml) in sterile 0.9% aqueous sodium chloride solution (Damytsia, Ukraine) and with 25 mΐ of 2 mM of chromogenic substrate S2238 (Chromogenix, Sweden) for 5 min at 37°C in a microplate well (MICROLON (Greiner Bio-One, Germany). The optical absorption is then determined using a microplate reader Multiscan EX (ThermoFisher Scientific, USA) at a wavelength of 405 nm. The optical absorption at a wavelength of 405 nm corresponds to the concentration of n- nitroaniline, which is split out of the chromogenic substrate S2238 by the action of thrombin formed from prothrombin by proteolysis with the enzymatic activator.

The results of the study are shown in the Table 8; the activity of the formed thrombin is expressed as a percentage, where the activity of thrombin provided by the initial solution of the enzymatic activator is taken for 100%.

Table 8

Relative amidolytic activity of thrombin, which provides the addition to blood plasma of the enzymatic activator of the blood coagulation system during its long term storage

Thus, the enzymatic activator in the lyophilized haemostatic agent does not lose its initial activity against the formation of thrombin, but retains it at the initial level during long-term storage at room temperature.

In view of the results given in the Table 8, it can be concluded that the lyophilized haemostatic agent obtained according to this invention has significant advantages over the ex tempore haemostatic agent according to the patent UA 117852 C2, namely it is more stable and retains the initial activity against the formation of thrombin during long-term storage. Example 6

Comparative analysis (control) of the sterility of the lyophilized haemostatic agent according to this invention and of the ex tempore haemostatic agent obtained by the method according to the patent UA 117852 C2 after a long storage time

The agent sterility control is carried out by the methods well known in this field, for example in accordance with the industry standard OST 42-21-2-85 “Sterilization and disinfection of medical devices. Methods, means and modes”, as described in detail above.

The control of sterility of the lyophilized haemostatic agent according to this invention, carried out immediately after sterilization, showed the absence of microorganisms when using sterilization by radiation with doses of 10, 15, 20 and 25 kGy, which indicates the sterility of the haemostatic agent according to this invention.

To check the sterility of the lyophilized haemostatic agent according to this invention, and of the ex tempore haemostatic agent obtained by the method according to the patent UA 117852 C2, for a long storage time a direct inoculation (immersion of the product without packaging, rinsing from the product without packaging) of standard nutrient media for sterility control, in particular thioglycollate medium (TITAN BIOTECH LTD, India), Saburo medium (LLC “Farmaktyv”, Ukraine), is performed. In a blind experiment the sterility of the agents, namely three samples of each, which were stored at 23 °C for 6 months at a relative humidity of not more than 25%, is compared.

Qualitative and quantitative analyses of sterility are performed. A sample is considered sterile if no more than lxl O ¹ CFU are detected in the sample. The results of the sterility study are shown in the Table 9. Table 9

Comparison of the sterility of the haemostatic agent according to this invention and the agent obtained according to the patent UA 117852 C2, when stored for 6 months.

Mesophilic aerobic and optionally anaerobic spore-forming bacteria were detected in the sample washings of the agent obtained according to the patent UA 117852 C2. Samples of the haemostatic agent according to this invention were sterile when stored for 6 months.

It is clear that the claimed invention is not limited to the following examples of specific implementation and may have numerous changes and modifications, and the scope of the invention rights is determined by the attached summary of invention. References Belozerskaya G.G., Makarov V.A., Aboiants R.K., Istranov L.L., Malykhina L.S., Zhydkov E.A. Application drug for haemostasis in case of capillary and parenchymal bleeding //Surgery. - 2004. - No. 9. - P. 55-59. Schonauer C, Tessitore E, Barbagallo G, Albanese V, Moraci A. The use of local agents: bone wax, gelatin, collagen, oxidized cellulose //Eur. Spine J. -2004. -JVb 6.-P. 36-40. Krylov Yu.F., Iziumov E.G., Gasanov M.T. Pharmacology of local haemostatic agents // International collection of research papers of the V research and practice conference on creation and approbation of new medicines “Medicines for a human”. - Kaunas. - 1997. - P. 87-94. Severtsev A.N., Brekhov E.I., Mironov N.P. et al. The use of local pharmacological agents to achieve final haemostasis during liver resections //Surgery. - 2001. - No. 1. - P. 86-90. QuikClot® use in trauma for hemorrhage control: case series of 103 documented uses /P. Rhee, C Brown, M. Martin [et al.] J. Trauma. - 2008. -Vol. 64, N4.-P. 1093-1099. Lawton, G. Novel haemostatic dressings /G. Lawton, J. Granvill-Chapman, PJ. Parker //J.R. Army Med. Corps. - 2009. - Vol. 155, N 4. - P. 309-314. QuikClot^® use in trauma for hemorrhage control: case series of 103 documented uses /P. Rhee, C. Brown, M. Martin [et al.] //J. Trauma. -2008.-Vol. 64, N4.-P. 1093-1099. Lawton, G. Novel haemostatic dressings /G. Lawton, J. Granvill-Chapman, PJ. Parker //J.R. Army Med. Corps. - 2009. - Vol. 155, N 4. - P. 309-314. Bordakov V.N., Doronin M.V., Rasiuk E.D. The efficacy of a new haemostatic agent “Fibrinostat” in the in vitro and in vivo experiments. //Medical journal. - 2009.- No. l. - P. 129-132. . Pozza, M. Celox (chitosan) for haemostasis in massive traumatic bleeding: experience in Afghanistan /M. Pozza, R.W.J. Millner //Eur.J. Emerg. Med. - 2OI L-V0I. 18, N l.-P. 31-33. . Wedmore I, McManus JG, Pusateri AE, Holcomb JB. A special report on the chitosan-based hemostatic dressing: experience in current combat operations // J Trauma. - 2006. - 60. - P. 655-658. . Kumar V.A., Wickremasinghe N.C., Shu S. and Hartgerink J.D. Nanofibrous snake venom hemostat // ACS Biomater. Sci. Eng., 2015. -1(12).-P. 1300-1305.. Krutikov M.G., Bobrovnikov A.E. Local treatment of wounds and bums //REMEDIUM. - 2006. - No. 5. - P. 41-42. . Feskov A.E., Sokolov A.S., Soloshenko S.V. A new haemostatic bandage based on the natural biopolymer of chitosan // Medicine of emergency. - 2017. - No. 2 (81). -P. 164-167. . Solovyov D.A., Platonova T.N., Ugarova T.P. Isolation and characteristic of ekamulin - prothrombin activator from the venom of Echis multisquamatus //Biochemistry. - 1996. -61. - 6. - P. 775-786. . Patent of Ukraine UA 117852 C2, 12.02.2018. . Certificate of registration for “Dressings based on fibre carbon-base materials” No. 9698/2010/. . Eretskaya E.V., Sakhno L.A., Nikolaev V.G. Application sorption: experience in clinical use and prospects of development //Biomater. Artif. Cells Immobilization Biotechnol. - 1991. - 19(1). - P. 129-145. . Patent for utility model No. 78585 U as of 10.04.2014 “The method of using a carbon-base sorbent dressing for medical purposes” Nikolaiev Y.H., Kudriachenko V.V., Kolosov A.Ye., Sakhno L.O., Sydorenko O.S. . Patent for utility model No. 92082 U as of 25.07.2014 “Dressing based on fibre carbon-base sorbents” Sniezhkova S.O., Sakhno L.O., Nikolaiev V.H. . Davydenko V.M. Radiobiology / V.M. Davydenko - Mykolaiv: Published by MSAU, 2011. - 265 p. . Bederson, Joshua B., Isabelle M. Germano, and Lorraine Guarino. “Cortical blood flow and cerebral perfusion pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat.” Stroke 26.6 (1995): 1086-1092. . Komisarenko S.V., Dieiev V.A., Luhovskoi E.V. and others. Methodical recommendations “Application of immunoenzymatic methods for diagnosing the threat of intravascular thrombosis”. - 2019. - “Publisher Bykhun V.Yu.”, - P.36.. Storozhuk OB, Panasenko OB, Storozhuk BG, et al. Wiad Lek. 2018;71(9): 1661 -1665. . Lugovskoi E.V., Kolesnikova I.N., Platonova TN, [et al.] Simultaneous quantification of soluble fibrin and D-dimer in blood plasma for the assessment of the threat of thrombosis. Klin Med (Mosk). 2013. 91, 11: 38-44.

Claims

1. The method of production of a haemostatic agent to control major bleeding, in case of haemophilia as well, comprising a dressing material and an active haemostatic component, in which the following stages are performed: a dressing material is prepared; the solution of an active haemostatic component - ekamulin is prepared; the solution of an active haemostatic component is applied to the prepared dressing material, and the active haemostatic component is non-covalently immobilized with a further freezing and lyophilizing to obtain a haemostatic agent; and the mentioned haemostatic agent is sterilized by radiation.

2. The method according to the clause 1 , in which activated fibre carbon-base material for medical purposes is used as a dressing material.

3. The method according to the clause 1 or 2, in which the preparation of the dressing material includes rinsing with distilled water, giving the required size and shape, and heat treatment.

4. The method according to the clause 3, in which the heat treatment is performed in a drying oven at a temperature between 80 and 120°C, preferably at a temperature of 90°C, for 1 -2 hours.

5. The method according to any of the clauses 1-4, in which the preparation of the solution of an active haemostatic component involves dissolving the lyophilisate of ekamulin in 0.9% aqueous sodium chloride solution containing the excipient calcium chloride in the amount of 10⁴-10³ M, to a final concentration of ekamulin of 0.7 - 1.0 pg/ml.

6. The method according to the clause 5, in which the preparation of the solution of an active haemostatic component additionally involves sterilization under aseptic conditions by passing through a sterilizing filter, preferably through a sterilizing filter with a pore size of 0.2 p .

7. The method according to any of the clauses 1 - 6, in which the solution of an active haemostatic component is applied in the amount of 8 ± 0.5 pg of ekamulin to 1 cm² of prepared dressing material.

8. The method according to any of the clauses 1 - 7, in which, after immobilization of an active haemostatic component, freezing is performed at a temperature of -20 ± 2°C for 12-15 hours.

9. The method according to any of the clauses 1 - 8, in which, after freezing, lyophilisation is performed at a condenser temperature of -83°C and a pressure of 0.05 millibars for 12 hours.

10. The method according to any of the clauses 1 - 9, which additionally includes packaging of the haemostatic agent in a sealed package before performing sterilization by radiation.

11. The method according to any of the clauses 1 - 10, in which the sterilization by radiation is performed with a radiation dose of up to 25 kGy.

12. The method according to any of the clauses 1 - 11, in which the haemostatic agent is an application haemostatic agent.

13. A haemostatic agent for major bleeding control, in case of haemophilia as well, is obtained under the method according to any of the clauses 1-12.