UA74045C2 - Suspension of fibrinogen, thrombin, and alcohol, method for its preparation, method for coating carrier, method for drying carrier coated with suspension of fibrinogen, thrombin, and alcohol, collagen sponge coated with suspension of fibrinogen, thrombin, and alcohol, use of coated collagen sponge for tissue gluing, tissue sealing and haemostasis - Google Patents

Abstract

A suspension of fibrinogen, thrombin, alcohol and optionally aprotinin is obtained by mixing fibrinogen in alcohol with thrombin in alcohol. The suspension contains fibrinogen and thrombin particles with a Folk Ward mean diameter of 25-100 m. The thrombin may be human, bovine or recombinant. The fibrinogen may be human or recombinant. A method for coating a carrier, such as a collagen sponge, with the suspension, and a method for drying the coating is disclosed. The coated collagen carrier may be used as a ready-to-use absorbable composition for tissue gluing, tissue sealing and haemostasis wherein the carrier is coaled with solidly fixed components of fibrin glue, i.e. fibrinogen and thrombin.

Description

Description of the invention

The invention relates to a suspension containing fibrinogen, thrombin, alcohol and, optionally, aprotinin. 2 In addition, the invention relates to a method of preparing such a suspension and a method of coating a carrier with such a suspension. The carrier can be a collagen carrier, for example, a collagen sponge. Further, the invention relates to a method of drying a coated carrier, in particular, to a collagen carrier coated with the proposed suspension, thereby obtaining a coated collagen carrier having solid active substances fixed on the carrier.

The coated collagen carrier can be used for tissue bonding, tissue closure, and hemostasis as a ready-to-use, absorbable composition consisting, preferably, of a carrier on which a coating of solid components of fibrin glue is fixed: fibrinogen and thrombin Such a fixed composition can be applied, for example, directly to the surface of the wound. When in contact with blood, total body water or physiological solution, the mechanism of this system mimics the 12 final stage of the coagulation system, in which thrombin catalyzes the conversion of fibrinogen to fibrin and the activation of factor XII into factor HIPA. The formed Xa factor stabilizes the fibrin clot by covalent cross-linking.

As when using any two-component glue, the surface of the wound and the carrier are glued together by polymerization. During this process, which lasts about 3-5 minutes, the proposed composition is preferably pressed against the surface of the wound. The components of the proposed composition are enzymatically decomposed within approximately 4-6 months after application.

Fibrin glues that simulate the final stage of the coagulation system are available on the market, consisting of a highly concentrated solution of fibrinogen, which is mixed with a thrombin solution before being applied to the surgical wound. These mixtures contain inhibitors of fibrinolysis, for example, aprotinin or ε-aminocaproic acid, to prevent premature dissolution of the fibrin clot by fibrolysin (plasmin). Such two-component glues (Ge) have a number of advantages when used in various surgical procedures, however, with severe bleeding, they can wash out before the bleeding is stopped. Further, these two-component adhesives require certain preparatory actions, including thawing or dissolving. Therefore, they are very impractical, work with them is burdensome, and for the successful use of these fibrin glues, you need to have experience. what

Over the past decade, surgeons have had the opportunity to choose from a large number of wound-healing fibrin adhesives for a number of indications. However, in most clinical trials using fibrin glues to improve hemostatic (hemostatic) properties and bonding properties, collagen was additionally used, which indicated their shortcomings and limited possibilities for use in surgery. hE

Collagen has been used as a hemostatic agent since the late sixties. Collagen is essential

With structural protein in all mammals. A monomeric protein with a mass of approximately 300 kilodaltons (tropocollagen) is covalently cross-linked in specific areas. Because of this, the mature protein is insoluble and forms characteristic fibrils with high tensile strength. Numerous subclasses of collagen are known, the most widespread of which is type I collagen - the main type of collagen found in skin, bone tissue, tendons and cornea. "

Collagen is a fibrous protein consisting of a triple helix approximately 29 Ohms long. Five such C7C10 triple helices (tropocollagen molecules) are arranged in a checkerboard pattern and form a microfibril with a diameter of approximately 3.5 nm. These microfibrils have polar and nonpolar segments that readily enter into specific inter- and intrafibrillar interactions. The microfibrils are packed into tetragonal lattices and form subfibrils with a diameter of about 30 nm. These subfibrils then assemble into a collagen fibril, the basic unit of connective tissue, which has a diameter of several hundreds of nm and observed through an optical microscope as a thin line. Collagen can be used as a wound closure material, possibly with a coating containing fibrin glue. Fibrin glues, which are combinations of fibrinogen, thrombin, and aprotinin, have been used for many years years find successful therapeutic use for gluing tissues and nerve fibers and for closing surfaces in cases of mild bleeding. One of the disadvantages of fibrin glues is that in the case of severe bleeding, the glue is usually washed off before sufficient polymerization of fibrin has taken place. To eliminate of this deficiency, surgeons began to manually apply liquid f ibrin glues on an absorbable carrier, for example, on a bundle of collagen fibers.

Despite the impressive success of this joint application, this method has not yet been widely used, which is due to some of its shortcomings. Yes, the preparation of the drug is relatively difficult, the application of the method requires experienced and qualified personnel, and in emergency cases, the drug

GF) cannot be used immediately because it takes 10-15 minutes to prepare. These factors stimulated the development of a fixed combination of a collagen carrier with a coating of solid fibrinogen, solid thrombin and solid aprotinin, known from EP 0059265.

The function of the collagen carrier is described in EP 0059265 and is mainly to absorb the coagulating drug with which it is coated and to add mechanical stability to it.

The product, which combines the hemostatic properties of fibrin glue with the advantages of collagen as a carrier, is developed and manufactured under the trademark TaspoSotrF. TaspoSotrF is a ready-to-use and easy-to-use combination of a collagen patch coated with the following active components of fibrin glue: human fibrinogen, bovine thrombin and bovine aprotinin. because TaspoSotya has been sold since the early 1990s by Musotei RNagta and has passed clinical trials in

in Europe for more than 2,500 patients. In addition, the product has passed the program of clinical examinations in Japan for more than 700 patients with very different indications, for example, in liver and lung resection, biliary tract operations, in spleen, kidney and pancreatic surgery, in ear, throat, nose surgery, in operative gynecology and cardiovascular surgery. TaspoSotroe has been found to be effective and safe.

During the conducted clinical examinations, no clinical complications associated with the use of TaspoSot were noted.

MO 97/37694 (Ittypo Egapse 5.A. company) describes example 4, when the use of the collagen product TaspoSotryae failed to stop bleeding, which led to death from blood loss, in contrast to 7/0 Stopping bleeding within 5 minutes, when a collagen product that did not contain thrombin and was prepared according to UMO 97/37694 was used.

MO 94/40033 highlights the disadvantages of the bovine thrombin used in TaspoSotri F. They are that the use of bovine thrombin and thrombin of other animal species can lead to the introduction of a dangerous viral infection and the possible transfer of cow diseases, for example, bovine spongiform encephalopathy.

US patent 05 6177162 B1 describes a device and a method for obtaining a material for closing and healing wounds. This device contains a container, in the lower part of which there are two perforated plates movable relative to each other, and the suspension in the specified container drips onto a carrier that moves relative to the container under its lower part.

The task of the invention is to create an improved suspension that can be used, for example, as a 2o coating for a collagen carrier to obtain a ready-to-use, absorbable composition for gluing and closing tissues and stopping bleeding. Another task of the invention is to create a method of preparing such a suspension. In addition, the task of the invention is to create an improved method of coating a carrier, for example, a collagen carrier, with a suspension containing fibrinogen and thrombin. Another task of the invention is to create a method of drying a wet coating from a suspension applied to a carrier to ensure reliable fixation of the coating on the carrier. In addition, the task of the invention is to create a covered collagen sponge with a coating of fibrinogen and thrombin, which effectively imitates the final stage of the coagulation system when the covered collagen sponge comes into contact with blood, body water or physiological solution. Further, the task of the invention is to create a coated collagen sponge with the specified coating, which has sufficient fixation of the coating on the collagen sponge, namely, satisfactory resistance to abrasion of the coating during mechanical action. According to the first task of the invention, a suspension containing fibrinogen, thrombin, and alcohol is proposed, and the method of obtaining the specified suspension includes: - preparation of a mixture of fibrinogen and alcohol; o preparation of a mixture of thrombin and alcohol; mixing a mixture of fibrinogen and a mixture of thrombin to obtain the specified suspension, and the specified "suspension contains particles of fibrinogen and thrombin with an average particle diameter according to Roik U/aga in the range of 25-100 μm, for example, 35-80 μm, 40-78 μm, 40-75 μm . deviation within 5 µKm, for example, -4 µm, -3.5 µm, 12 µm, -1.5 µm, µm, -0.8 µm, -0.b µm, 40.5 µm. Such a suspension, when applied to a carrier such as a collagen carrier, has been found to be effective in a ready-to-use, "absorbable" composition for tissue sealing, tissue closure, and bleeding control. This suspension can optionally contain aprotinin added to the fibrinogen mixture in the form of a concentrated aqueous solution. As a dye, riboflavin can be added in such a way that the suspension applied to the carrier and dried can be easily identified.

In view of the physical properties of the suspension, especially its behavior from the point of view of settling of rather large particles in alcohol, it is impossible to apply any standard index of liquid viscosity of the suspension. In connection with this, an alternative method of ensuring the viscosity index was used. In this way, the suspension can have such a viscosity that 90-120 ml of the suspension flows only under the influence of gravity through the lower opening of the container, which has e - a cylindrical part with an inner diameter of 40-50 mm and an inner length of 55-65 mm, and av) - a conical part the lower part with a height of 17-23 mm with a lower hole at the lower end of the specified conical part in the form of a round hole with a diameter of 2 mm, in 25-75 seconds. If the container is made of steel, then the container and holes have the following dimensions: sl - the inner diameter of the cylindrical part: 4 mm; - internal height of the cylindrical part: bOhm; - inner height of the conical bottom: 20.5 mm; - inner diameter of the lower hole: 2.bmm; - length of the channel connected to the lower hole: Umm, or o if the container is plastic, then the container and holes have the following dimensions: ko - inner diameter of the cylindrical part: 50mm; - internal height of the cylindrical part: 41mm; 60 - inner height of the conical bottom: 24mm; - inner diameter of the lower hole: 2.5 mm; - the length of the channel connected to the lower opening: less than 5 mm.

The specified time for the suspension to flow can be within 25-60 seconds, for example, 25-50, 30-50, 32-44, 34-38 seconds. 65 The parameters and distinctive features of the suspension described below in connection with the second method according to the second task of the invention also apply to the suspension according to the first task of the invention.

According to the second task of the invention, a method of preparing a suspension of fibrinogen and thrombin is proposed, which includes: - preparing a mixture of fibrinogen and alcohol; - preparation of a mixture of thrombin and alcohol; - mixing of a mixture of fibrinogen and a mixture of thrombin to obtain the specified suspension, and the specified suspension contains particles of fibrinogen and thrombin with the average particle diameter according to Roik

UMUaga in the range of 25-100μm. The parameters and distinctive features described above in connection with the suspension according to the first task of the invention also apply to the method according to the second task of the invention. 70 At the stage of preparation of the fibrinogen mixture in a suitable way, for example, by passing through a sieve, it is possible to pre-select small particles of fibrinogen with a Roik Uuaga diameter in the range of 25-100 μm. In addition, at the stage of preparation of the specified mixture, fibrinogen can be directly homogenized in alcohol, preferably at a temperature in the range of 0-122C, for example, in the range of 2-82. During homogenization, the temperature can be lowered. The stage of mixing the mixture of fibrinogen and the mixture of thrombin can be carried out by stirring the suspension, and 75 can be mixed at a temperature in the range of 0-122C, for example, in the range of 2-826.

The thrombin may be human, bovine, or recombinant thrombin, and the fibrinogen may be human or recombinant fibrinogen. The alcohol can be organic, for example, methyl, ethyl, propyl, isopropyl, for example, anhydrous organic alcohol, anhydrous ethyl, anhydrous propyl or anhydrous isopropyl alcohol. Human fibrinogen can be supplied lyophilized in solid form.

In accordance with the third task of the invention, a method of applying a coating from a suspension containing fibrinogen and thrombin to the carrier is proposed, which is characterized by the fact that the suspension is prepared in a way that includes: - preparation of a mixture of fibrinogen and alcohol; - preparation of a mixture of thrombin and alcohol; - mixing of a mixture of fibrinogen and a mixture of thrombin to obtain the specified suspension, and the specified suspension contains particles of fibrinogen and thrombin with the average particle diameter according to Roik

UUaga in the range of 25-100μm, and the indicated coating method includes: - delivery of a suspension of fibrinogen, thrombin and alcohol to a place next to the carrier; - application of the specified suspension to the surface of the carrier to be covered. i am

The carrier can be a collagen carrier, for example, a collagen sponge.

A collagen sponge must meet at least one, and preferably several, of the following criteria: M - pH value: 5.0-6.0; o - lactic acid content: no more than 5905; - ammonium content: no more than 0.595; M - soluble protein content, calculated as albumin content: no more than 0.595; h- - sulfated ash content: no more than 1.095; - content of heavy metals: no more than 20 million"; - microbiological purity: no more than 1 OZKUO/G; " - collagen content: 75-10090; - density: 1-10 mg/cm3; c) c - modulus of elasticity: 5-100N/ cm7. z» The collagen sponge can be produced in a method consisting of the following stages: - preparation of the collagen gel; - adding air to the collagen gel and stirring to obtain the collagen foam; 35 - drying the collagen foam to obtain a dry block. collagen sponge having inside the chamber; 7 - separation from the collagen sponge block of sponge parts with a diameter of chambers greater than 0.75 mm and less than 4 mm g" or chambers with a diameter, on average, no more than Zmm.

In this context, the term "diameter of the chamber" should be understood as the greatest distance in a straight line between the walls of the chamber, that is, the greatest diagonal distance of the chamber in a straight line. Cameras can have a polygonal shape, for example, octagonal. It has been established that when the diameter of the chambers is greater than 0.75 mm and less than 4 mm or the diameter of the chambers does not exceed 3 mm on average, the collagen sponge is especially suitable for covering it with a suspension preparation containing fibrinogen and thrombin. It has also been found that a coated collagen sponge made in this manner is air- and moisture-tight in the sense that, when applied to a wound, it does not allow air or liquid to seep through the collagen sponge. 25 The suspension can be applied to the carrier at an ambient temperature in the range of 0-122C, for example, 1-102C, 2-896,

GF) Next, the suspension can be applied to the carrier at a relative humidity of the surrounding atmosphere in the range of 75-99905,

GI, for example, 75-9995, 85-9595, In the preferred version of implementation by 1 cm? 0.08-0.12 ml of suspension is applied to the surface to be covered. In order to ensure the same effectiveness of the finished carrier with the coating applied over the entire surface, the suspension is preferably evenly distributed over the given surface to be covered, with such a calculation that the mass of fibrinogen per unit area of the surface to be covered does not exceed 2596, for example, does not exceed 20, 15, 10905.

An applicator with at least one nozzle can be used to apply the suspension to the carrier.

A jet applicator under pressure supplies the suspension through a nozzle when the carrier and nozzle are moved relative to each other. The applicator may include a belt conveyor, an agitation device connected to or adjacent to a pump or pump system or other delivery equipment, and a nozzle or nozzle system moving transversely, for example at right angles to the belt conveyor. . Depending on the specific characteristics of the environment, the nozzle or nozzle system can have different shapes and sizes. A nozzle or a system of nozzles can be connected to the supply equipment with tubes. Feed equipment can provide coating media from the mixing device to the nozzle system. During the coating process, the nozzle system can be moved across the carrier. In the standby position, it can be kept away from the belt conveyor. The coating process can be started by a light sensor signal that detects the presence of a carrier on the conveyor belt, and can also be stopped by a light sensor signal. Such an applicator provides a relatively small 7/0 dead space, is easy to handle and easy to clean. Further, it provides the possibility to interrupt the coating process at any time, it can be used in a relatively wide range of viscosity values, and it provides a uniform coating.

As an alternative to the above method or in addition to it, an applicator can be used to apply the suspension to the carrier, with a container having several separate outlets, and the suspension from the container under /5 pressure is fed through the outlet openings to the carrier. The latest type of applicator in the form of a container with several movable plates on its bottom is described in US patent Mob177126 VI, which is incorporated into this application by reference. In view of the uniform distribution provided by the devices according to the US patent Mob177126

VI, one of these devices is used in a preferred embodiment of the present invention. When applying the suspension to the carrier, it and the applicator preferably move relative to each other in the direction of supply, and the speed of movement can be within 0.025-0.05m/s, for example, 0.03-0.04m/s. The consumption of the suspension applied through the applicator to the carrier can be in the range of 400-600 ml/min., for example, 470-550, 495-505 ml/min.

According to the fourth task, the invention relates to a method of drying a suspension of fibrinogen, thrombin and alcohol applied as a wet coating on the surface of the carrier to be covered. The specified method includes the stage of impacting the coated carrier with a pressure below 1000 mbar to obtain a dried surface of the carrier, the surface to be coated, and fixation of the dried coating to the surface to be coated. When using a vacuum for drying, it is possible to maintain a low temperature (2-102C) and a high relative humidity (80-9595), thanks to which i) it is possible to ensure the preservation of the structure and physical properties of the carrier, in particular, the carrier in the form of collagen, for example, a collagen sponge, and as well as fibrinogen and thrombin.

The suspension can be obtained by: u - preparation of a mixture of fibrinogen and alcohol; preparation of a mixture of thrombin and alcohol; - mixing of a mixture of fibrinogen and a mixture of thrombin to obtain the specified suspension, and the carrier can be a collagen sponge, made in a method consisting of the following stages: o - preparation of a collagen gel; - adding air to the collagen gel and mixing to obtain collagen foam; C - drying of the collagen foam to obtain a dry block of collagen sponge, which has a chamber inside; che - separation from the collagen sponge block of parts of the sponge with a chamber diameter greater than 0.75 mm and less than 4 mm or with a chamber with an average diagonal size of Zmm, and the coating can be applied to the collagen sponge by - delivering a suspension of fibrinogen, thrombin and alcohol to a place next to collagen sponge; "- applying the specified suspension to the surface to be covered with a collagen sponge. 8 c The methods of making a collagen sponge and preparing a suspension are discussed above in connection with the methods and in accordance with the second and third objectives of the invention. "" The carrier with the applied coating can be dried under the indicated pressure at a temperature within 0-12 es, for example, 1-1092С, 2-89 and (or) relative humidity of the surrounding atmosphere within the range of 75-9995, for example, 85-9595. When drying the carrier with the applied coating, you can blow with a stream of air to remove steam from the carrier. - and To complete the drying of the carrier with the applied coating, it is preferable to keep it under the specified conditions for not less than 1 hour, for example, not less than 2, not less than 4 hours.

Due to shrinkage, the area of the dried surface to be covered is smaller than the area of the wet surface to be (av) covered. In the proposed method, the area of the dried surface to be covered is at least 7595 out of 50 of the area in the wet state, in particular, at least 80905.

To ensure the stability of the active components during storage of the coated carrier, the moisture content of the carrier layer and the dried coated surface preferably does not exceed 1295 by weight, for example, does not exceed 895 by weight.

Any parameters and distinguishing features of the suspension and collagen sponge, including methods of their preparation, considered in connection with other tasks of the invention, also apply to the method according to the fourth task of the invention. o In accordance with the fifth task of the invention, a covered collagen sponge with a coating of fibrinogen and thrombin is offered, which is characterized by the fact that the covered collagen sponge is manufactured in a way that includes the following stages: - production of a collagen sponge in a way that includes: because - preparation of collagen gel; - adding air to the collagen gel and mixing to obtain collagen foam; - drying of collagen foam to obtain a dry block of collagen sponge contained inside the chamber; - separation from the collagen sponge block of sponge parts with a diameter of chambers greater than 0.75 mm and less than 4 mm or with chambers with an average diagonal size of Zmm; 6E - applying a suspension of fibrinogen, thrombin and alcohol to the surface of the collagen sponge to be covered, and - applying a pressure below 1000 mbar to the carrier with the coating applied, with such a calculation as to obtain a dried surface of the carrier to be covered and fixing the dried coating to the surface to be covered , and the collagen sponge with the applied coating has at least one of the following properties: - the suspension is evenly distributed over the given width of the surface to be covered, so that the mass of fibrinogen per unit area of the surface to be covered does not exceed 2590; - when shaking a sample of the material with the applied coating in the apparatus for shaking Mirgoiykh at a rotation frequency of approximately 800-1200 rpm. within 2 minutes, the abrasion of the coating is less than 2.Omg/cm 7,

Uniform distribution of the suspension over the surface to be covered increases the effectiveness of the specified surface when it is used, for example, for gluing and closing tissues or hemostasis. Due to the low attrition rate of the coating 70, the coated collagen sponge is portable, the surgeon can pick it up with hands and/or a surgical instrument and otherwise manipulate it without damaging the dried suspension, i.e. the coating. The fibrinogen compound content may be approximately 60-90% of the total weight of the coated collagen sponge. Said compound usually contains about 50-6095 by weight of the following substances: salts, amino acids and albumin. The content of fibrinogen itself in the composition is usually 40-50905.

The moisture content in the suspension is preferably in the range of 20-8Omg/ml, for example, 24-32mg/ml. Thrombin content can be in the range of 20-40 IU/ml, for example, 24-33 IU/ml. On average, the thrombin content after coating can be in the range of 2-41 IU/cm?, for example, 2.3-3.3 IU/cm2. Can it be desirable that the content of thrombin on any part of the coated surface does not exceed 5 IU/cm? or not to exceed 3.8 IU/cm3.

The microbiological purity of the carrier with the applied coating is preferably no more than 4 CFU/cm?, in particular, no more than 2.2 CFU/Icm?.

In the next independent aspect, the invention relates to the use of the specified collagen sponge with an applied coating for gluing and closing tissues and hemostasis.

Figures 1-7 show various coated media and tools for their application, as described in the MIP example. with

Fig. 8 is a block diagram illustrating the operations from preparation of suspension to packaging of collagen Ge) sponge with applied coating.

Fig. 9 illustrates the devices used to measure the viscosity index of the suspension.

Fig. 10 and 11 are a block diagram illustrating the process of manufacturing a collagen sponge.

Below is a description of preferred embodiments of the proposed methods and products (see also yuyu

Fig.B8). "

A suspension containing fibrinogen, thrombin and alcohol can be prepared in the proposed method of preparation of the suspension, namely: -

Fibrinogen is homogenized in 10096 ethyl alcohol at 2-83 and a mixture of fibrinogen and alcohol is obtained, and this mixture is approximately 8095 of the final volume of the suspension. Then riboflavin is added.

After that, the mixture is stirred in a closed vessel until the next stage of working with it. -

Human thrombin or bovine thrombin is diluted with water for injection. The resulting solution at 0-83 is added to a 35-fold volume of 10095% ethyl alcohol. The thrombin suspension obtained in this way is homogenized at 0-820 for 80-100 seconds. " 20 Before mixing the mixture of fibrinogen and thrombin, a solution of aprotinin and water for injections are added to the mixture of fibrinogen. Then the thrombin mixture is added to the fibrinogen mixture. By adding the 10095th. of ethyl alcohol, the volume c of the suspension is brought to the final condition. :z» The methods, which include the stages described above, are referred to as "group I methods" in the text below.

As an alternative, the method of preparation of the proposed suspension containing fibrinogen, thrombin and alcohol consists of the following stages: -1 The fibrinogen mixture is obtained by adding, during mixing, pre-selected small particles of fibrinogen with an average diameter according to Roik Uuaga in the range of 35-8μm and riboflavin in 94- 9796th ethyl alcohol at 2-8°C. The mixture of riboflavin and ethyl alcohol obtained in this way is approximately 70-8090 of the final volume of the suspension. After that, the fibrinogen mixture is stirred in a closed vessel until further operation. p The thrombin mixture is obtained by adding thrombin to 94-9795% ethyl alcohol at -302C. The resulting sp thrombin mixture is homogenized for 80-100 seconds. In another embodiment, the thrombin mixture is obtained by dissolving thrombin in water for injections, after which the resulting thrombin solution is slowly added to a 35-fold volume of 100965% ethyl alcohol at -302C. The resulting suspension is homogenized for 80-100 seconds.

As equipment for homogenization, you can use OrigaTitakh of the IKA company.

The thrombin mixture is added to the mixture containing fibrinogen and riboflavin. Then add 94-9796 ethyl alcohol at 2-826. The methods that include the stages described above are referred to as "group II methods" in the text below.

In the methods of group I and group II, it is possible to obtain the proposed suspension mainly with the following characteristics: 60 - ethyl alcohol content: 94-9790; - leakage time measured by the device shown on the left in Fig. 9: 31.5-48 seconds; - deposition characteristics (volume of solid particles as a percentage of the total volume): 5 minutes after the start of the exam: more than 8590; 24 hours after the start of the exam: 50-8090; - size of particles: average diameter according to Roik UUaga 35-8Ωm. 65 In one of the preferred variants of the method of drying, strips of collagen sponge are incubated at 2-82 and relative humidity of 80-9195 for 2-30 hours before coating the carrier in the form of collagen sponge. The applicator for applying the suspension to the collagen sponge is described above. The coated collagen sponge strips are incubated for 8-60 minutes at 2-8 2С and relative humidity of 80-9095. Then the coated collagen sponge strips are dried in a vacuum drying chamber at an air temperature of 2-89 and a relative humidity of 80-9095. At the same time, a flow of air with a flow rate of 1.2-40 m3/h is passed through the respiratory valve above the collagen strips. A 30-bOmbar vacuum is created, that is, an absolute pressure of approximately 97Ombar, depending on the atmospheric pressure, and the collagen strips with the applied coating are dried for 2-5 hours.

The specified viscosity of the suspension is obtained using one of the devices shown in Fig.9 and described above.

The device shown on the left in Fig. 9 is made of steel, while the container and the lower opening have the following dimensions: - the inner diameter of the cylindrical part: 4 mm; - internal height of the cylindrical part: bOhm; - inner height of the conical bottom: 20.5 mm; - inner diameter of the lower hole: 2.bmm; - the length of the channel connected to the lower opening: Umm.

The device shown on the right in Fig. 9 is made of plastic, and the container and the bottom hole have the following dimensions: - inner diameter of the cylindrical part: 5 Ωm; - internal height of the cylindrical part: 41mm; - inner height of the conical bottom: 24 mm; - inner diameter of the lower hole: 2.5 mm; - the length of the channel connected to the lower opening: less than 5 mm. sch o porous natiya 87400084 you are with her you 00000010 592

Pstdia 0001000 350 o aka 10012 - i - « t z s g

The I-MI examples below illustrate various procedures for manufacturing a coated collagen sponge with the proposed fibrinogen and thrombin coating. These procedures include the proposed methods and preparation of the suspension, resulting in the implementation of the proposed suspension in the variants described below. In these driving examples, the proposed coating and drying methods are used.

Example o In this example, the suspension contains human fibrinogen of composition B and human thrombin of composition B. 50 of them. The final volume of the suspension, 3500 ml, was obtained by the method of group II using the following quantities and parameters:

Fibrinogen mixture: sl - 2800 ml of ethyl alcohol (9495 at 2-82С); - 492.5g of fine human fibrinogen composition B; - 493.5 mg of riboflavin. 59 The fibrinogen mixture was stored for 20 hours at 2-82C with stirring.

F! Thrombin mixture: - AMb0ml of ethyl alcohol (10095 at -302С); o - 12.27 g of human thrombin composition B.

The thrombin mixture was stored for 18 hours at -3020. bo Suspension: - 157ml of thrombin mixture was added to the fibrinogen mixture; - by adding 9495% ethyl alcohol at 2-82C, the volume of the suspension was brought to the final volume of 3500 ml.

Characteristics of the suspension: 1. Concentration of ethyl alcohol: 94.3905. 65 2. Leakage time measured in the steel device shown on the left in Fig. 9: 36.5 seconds.

3. Deposition: a) volume of deposition 5 minutes after the start: 98905 of the test volume; b) deposition volume 24 hours after the start: 6495 test volume. 4. Size of particles (average diameter according to Roik UMaga): 56.5--1.3 μm.

A carrier in the form of strips of collagen was coated with a suspension. First, 48 collagen sponge strips were pre-incubated in a cooling chamber under the following conditions: - temperature: 5.290; - absolute humidity: 4.8 g of water per kg of air; - incubation time: 18.5 hours.

The applicator described in the US patent was used to coat the collagen sponge strips with a suspension

Mob 6177126 B1.

The coated collagen sponge strips were dried as follows:

Strips with the applied coating were incubated for 15 minutes at a temperature of 5.22C and an absolute humidity of 75 4.8 g of water per kg of air.

Then the coated strips were dried in a vacuum drying chamber under the following drying conditions: - air: temperature 5.22C, absolute humidity 4.8g of water per kg of air; - air consumption through the breathing valve: 2SmU/h; - vacuum: 5Ombar; - drying time: 4 hours.

Erasure of the resulting coating on strips of collagen sponge when shaking a 1-hosm sample in a test tube in a Migboi shaking machine at a rotation frequency of 800-1200 rpm. within 2 minutes was approximately 0.2 mg/cm7.

Example II p

In this example, the suspension contains human fibrinogen of the composition C and human thrombin of the composition C Ge)

The final volume of suspension Z3500ml was obtained by the method of group II using the following quantities and parameters:

Fibrinogen mixture: - 2252 ml of ethyl alcohol (9495 at 2-82C); - 370.7 g of fine human fibrinogen composition C; yuyu - 493.5 mg of riboflavin. hE

The fibrinogen mixture was stored for 20 hours at 2-82C with stirring.

Thrombin mixture: o - 1 ml of ethyl alcohol (10095 at -302С); "I - 12 ampoules of human thrombin composition C (10650 IU/ampoule)/12 ml of water for injections. M

The thrombin mixture was stored for 18 hours at -3020.

Suspension: - 164.5 ml of thrombin mixture was added to the fibrinogen mixture; - by adding 9495% ethyl alcohol at 2-82C, the volume of the suspension was brought to the final volume of 3500 ml. "

Characteristics of the suspension: with c 1. concentration of ethyl alcohol: 94.1905. 2. the leakage time measured in the steel device shown in Fig. 9: 32.8 seconds. from 3. deposition: a) volume of deposition 5 minutes after the start: 94905 of the test volume; b) deposition volume 24 hours after the start: 7195 test volume. -I 4. Particle size (average diameter according to Roik UMaga): 49.2--0.93μm.

A carrier in the form of strips of collagen was coated with a suspension. First, 48 strips of collagen sponge were pre-incubated in a cooling chamber under the following conditions: o - temperature: 4.890; - relative humidity: 90.390; shk - incubation time: 22.25 hours. sl The applicator described in the US patent was used to cover the collagen sponge strips with a suspension

Mob 6177126 B1.

The coated collagen sponge strips were dried as follows:

The coated strips were incubated for 13 minutes at a temperature of 4.99C and an absolute humidity of 4.8 g of water per kg of air.

Then the coated strips were dried in a vacuum drying chamber under the following drying conditions: co - air: temperature 5.22C, absolute humidity 4.9g of water per kg of air; - air consumption through the breathing valve: 25 MmZ/h; bo - vacuum: bOmbar; - drying time: 4 hours.

Erasure of the resulting coating on strips of collagen sponge when shaking a 1-hom sample in a test tube in a Migboi shaking machine at a rotation frequency of approximately 800-1200 rpm. within 2 minutes was approximately 0.Zmg/cm. because Example PI

In this example, the suspension contains human fibrinogen composition B and human thrombin composition B and aprotinin.

The final suspension volume of 10000 ml was obtained by the method of group II using the following quantities and parameters:

Fibrinogen mixture: - 820ml of ethyl alcohol (100905 at 2-82), 39.4ml of water for injections and 10.bml of aprotinin; - 90.67 g of fine human fibrinogen composition B; - 141 mg of riboflavin.

The fibrinogen mixture was stored for 20 hours at 2-82C with stirring.

Thrombin mixture: - BOoml of ethyl alcohol (10095 at -302С); - 3.75 g of human thrombin composition B.

The thrombin mixture was stored for 16 hours at -3020.

Suspension: - the entire volume of the thrombin mixture was added to the fibrinogen mixture; - by adding 10095% ethyl alcohol at 2-82C, the volume of the suspension was brought to the final volume of 1000 ml.

Characteristics of the suspension: 1. concentration of ethyl alcohol: 9590. 2. flow time measured in the plastic device shown on the right in Fig. 9: 35 seconds. 3. deposition: a) volume of deposition 5 minutes after the start: 89905 of the test volume; b) deposition volume 24 hours after the start: 7695 test volume. 4. Particle size (average diameter according to Roik UMaga): 74.4-3.5 mm.

A carrier in the form of strips of collagen was coated with a suspension. First, 16 strips of collagen sponge were pre-incubated in a cooling chamber under the following conditions: c - temperature: 5.02C; o - relative humidity: 8590; - incubation time: 17 hours.

The applicator described in the US patent was used to coat the collagen sponge strips with a suspension

Mob 177126 VI. at

Strips of collagen sponge with the applied coating were dried in the following way: "g

Strips with the applied coating were incubated for 35 minutes at a temperature of 5 2С and a relative humidity of 859. o

Then the coated strips were dried in a vacuum drying chamber under the following drying conditions: "I - air: temperature 52C, relative humidity 8590; 35 . - , with - - air consumption through the breathing valve: 1.2 m7/h; - vacuum: 35mbar; - drying time: 4 hours.

Abrasion of the resulting coating on collagen sponge strips when shaking a 1 x 5 cm sample in a 20" test tube in a Migboi shaking machine at a rotation frequency of approximately 800-1200 rpm. within 2 minutes was approximately 0.2 mg/cm3.

An example for THEM;" In this example, the suspension contains human fibrinogen of composition C and human thrombin of composition C.

The final suspension volume of 78 Oml was obtained by the method of group II using the following quantities and parameters:

Fibrinogen mixture: - I - 70 Oml of ethyl alcohol (9495 at 2-82); - 84.42 g of fine human fibrinogen composition C; t - 110 mg of riboflavin. o The fibrinogen mixture was stored for 20 hours at 2-82C with stirring.

Thrombin mixture: ve - ZBml of ethyl alcohol (10095 at -302С); sl - 0.54 g of human thrombin composition C.

The thrombin mixture was stored for 16 hours at -3020.

Suspension: - 23.00 ml of thrombin mixture was added to the fibrinogen mixture; o - by adding 10095% ethyl alcohol at 2-82C, the volume of the suspension was brought to the final volume of 780 ml.

Suspension characteristics: where 1. concentration of ethyl alcohol: 9490. 2. flow time measured in the plastic device shown on the right in Fig. 9: 33.5 seconds. 60 2. deposition: a) volume of deposition 5 minutes after the start: 92905 of the test volume; b) deposition volume 24 hours after the start: 7295 test volume. 3. Particle size (average diameter according to Roik U/aga): 60.5--0.5 μm.

A carrier in the form of strips of collagen was coated with a suspension. First, 8 strips of collagen sponge were pre-incubated in a cooling chamber under the following conditions:

- temperature: 6.02; - relative humidity: 8590; - incubation time: 18.5 hours.

The applicator described in the US patent was used to coat the collagen sponge strips with a suspension

Mob 6177126 B1.

The coated collagen sponge strips were dried as follows:

The coated strips were incubated for 45 minutes at a temperature of 52C and a relative humidity of 8590. Then the coated strips were dried in a vacuum drying chamber under the following drying conditions: - air: temperature of 52C, relative humidity of 8590; - air consumption through the breathing valve: 1.2 mZ/h; - vacuum: 35mbar; - drying time: 4 hours.

Abrasion of the resulting coating on collagen sponge strips when shaking a 1-hom size sample in a test tube in a Mirgoiich shaker at a rotation frequency of approximately 800-1200 rpm. within 2 minutes was approximately 0.Zmg/cm?.

Example M

In this example, the suspension contains human fibrinogen composition A and human thrombin composition A.

The final suspension volume of 3120 ml was obtained by the method of group I using the following quantities and parameters:

Fibrinogen mixture: - 2540 ml of ethyl alcohol (10095 at 2-82C); - 311.6 g of human fibrinogen composition A; - 4A4Omg of riboflavin. with

The fibrinogen mixture was stored for 18 hours at 2-82C with stirring. Gee)

Thrombin mixture: - 210 ml of ethyl alcohol (100 95 at -302С); - 229g of human thrombin composition A. yu

Suspension: - 87.3 ml of water for injections was added to the fibrinogen mixture; "And - a mixture of thrombin was added to the fibrinogen mixture; o - by adding 10095% ethyl alcohol at 2-82C, the volume of the suspension was brought to the final volume.

Characteristics of the suspension: "And 1. concentration of ethyl alcohol: 9790. 2. time of outflow, measured in the steel device shown on the left in Fig. 9: 40.8 seconds. 3. deposition: a) deposition volume 5 minutes after the start: 95.6905 of the test volume; b) deposition volume 24 hours after the start: 63.595 test volume. « 20 4. Particle size (average diameter according to Roik UMaga): 51.80, microns. -о с The carrier in the form of strips of collagen was coated with a suspension. First, 48 strips of collagen sponge were pre-incubated in a cooling chamber under the following conditions: :z" - temperature: 6.590; - relative humidity: 9090; - incubation time: 22.5 hours. -And the applicator disclosed in the US patent was used to cover the collagen sponge strips with a suspension

Mob 6177126 B1. o The coated collagen sponge strips were dried as follows: o The coated strips were incubated for 10 minutes at a temperature of 6.52 and a relative humidity of 5°C.

Then the coated strips were dried in a vacuum drying chamber under the following drying conditions: 4 - air: temperature 6.52C, relative humidity 9090; - air consumption through the breathing valve: 21 mU/h; - vacuum: 5v8mbar; - drying time: 4 hours.

Erasure of the resulting coating on strips of collagen sponge when shaking a 1-hom sample in a test tube in a Migboi shaking machine at a rotation frequency of approximately 800-1200 rpm. within 2 minutes was approximately 0.2 mg/cm?.

Example MI 60 In this example, the suspension contains human fibrinogen composition A and bovine thrombin and aprotinin.

The final suspension volume of 16720 ml was obtained by the method of group | using the following quantities and parameters:

Fibrinogen mixture: - 13,600 ml of ethyl alcohol (10,095 at 2-82); - 1750.5 g of human fibrinogen composition A; because - 2361 mg of riboflavin.

The fibrinogen mixture was stored for 21 hours at 2-82C with stirring.

Thrombin mixture: - 420 ml of ethyl alcohol (10095 at -302С); - 1229g of bovine thrombin.

Suspension: - 162 ml of aprotinin solution was added to the fibrinogen mixture; - 304.7 ml of water for injections was added to the fibrinogen mixture; - a mixture of thrombin was added to the fibrinogen mixture; - by adding 10095% ethyl alcohol at 2-82C, the volume of the suspension was brought to the final volume.

Characteristics of the suspension: 1. concentration of ethyl alcohol: 9790. 2. flow time measured in the steel device shown on the left in Fig. 9: 36.8 seconds. 3. particle size (average diameter according to Roik U/aga): 58.6--O0.bmcm.

A carrier in the form of strips of collagen was coated with a suspension. First, 288 collagen sponge strips were pre-incubated in a cooling chamber under the following conditions: - temperature: 6.590; - relative humidity: 89905; - incubation time: 25 hours.

The applicator described in the US patent was used to coat the collagen sponge strips with a suspension

Mob 6177126 B1.

The coated collagen sponge strips were dried as follows:

Strips with the applied coating were incubated for 10 minutes at a temperature of 6.52 and a relative humidity of 899.s

Then the strips with the applied coating were dried in a vacuum drying chamber under the following drying conditions: o - air: temperature 6.52C, relative humidity 8990; - air consumption through the breathing valve: 22.5 mZ/h; - vacuum: 5Ombar; - drying time: 4 hours. at

Abrasion of the obtained coating on strips of collagen sponge when shaking a sample of 1 hbsm size in the "Ж test tube" in an apparatus for shaking Mibgoiykh at a rotation frequency of approximately 800-1200 rpm. within 2 minutes was approximately 0.2 mg/cm?.

Example of MY "I

A collagen sponge with a coated coating containing human fibrinogen, bovine thrombin and aprotinin, M, investigated the stability of the coating suspension during its application for 7 hours in the conditions of industrial premises, for example, at 2-826.

Samples of each active ingredient were taken at different times. The results are given in the table. « и - 22222000 Ioeryyuoyunludyaya semlu chenlu tyumen (momludprotiniya (units veremnl) s :» 75 The results indicate high stability of all three components.

An example of MIA with" The description is carried out with reference to Fig. 1-7, where: o Foot

Ф» 70 ploorvkov bezpoyuyt/opporytyamo 1111111 on the stack oryaeknvporytyam priv dlyandiyut 11111111

Тл оревнбзпоюипямурузурнуюмумулиади after inserting from the device for ndosyut 11111111

F

With holes without coating with slags 0 85 and with slags: insert t for zndozhot 11111111

SU 00 rzunovryuyuolezkrytuyam urozornuy vytkavyam after inserting from the pristy for so far 11111111 yuyu Fiuvaz

From Europe Europe without coating/coating 32 ovrot rev with coating: insertion into the device for endoscoly 11 77 Europe With the coating of the broken hood after insertion into the device for endosylation 0000

Fyuvaya al vyvove ayu bezpoitya merge 11111111 for vnkote Ryu tokrtyayu: insert tu dlyazndozht 11111111 dv With Envote Reyut cover urozornuyu zytaya after inserting into the device dai sndosyut 11111111

Figure 5

BI tart Mo kly bezpofitya zlotyyam 52 tarovtr MO Ki coated insert device for dos 111 53 Taroztre MOKpyzpokritiyam: in unfolded form after insertion in the device for endoscotia 00 iovuvav 51 rode Musoten bezpoytya, stokram borrotatov zhayuyu 11111111 52 sponge Musote coated (laboratory sample) insert in prissyute for n 11111111 6.3 Musotes collagen sponge with coating (laboratory sample); in the unfolded form after insertion into the device for endoscopy 53 Collagen sponge Musotech with coating (production sample - Taspobotv); In the unfolded extension of the calf, inserts in the endoscope port. yuoyayat 00 p zhtTtTtTtTttTttTttTttnnnnnshshni

Intument for endoscopy vloozkO 11111111

Pnstrument for endoscopy Epoobosky 0

Comparison of Musoted coated sponge (TaspoSot 5) with other carrier products with a coating similar to TaspoSot 5.

Adhesion layer

Method 1. Applying a coating to various media

ESchizogye Raisn PolyglactinU10/polydiaxanone | oppwop/doppzop(manufacturer) Germany, 22851 Norderstedt, pom Ind My o cellulose Robert-Koch-strasse, 1

Musotei Io)

A suspension of TaspoSot 5 was applied as a coating to the surface measuring 2x4.5 cm of each carrier. The amount of the suspension corresponded to the technical conditions of TaspoSot (5.5 mg of fibrinogen/cm). The samples were dried. o 2. A 1x4 sample of each carrier with an applied coating was prepared. " 3. The adhesion of the layer was tested as follows:

З Description of the method in.

Equipment

Analytical scales (measurement accuracy 0.5 mmHg)

A device for shaking Mibgoiyh in combination with a fixation device "

Ruler graduated in millimeters

A stopwatch, a scalpel, tubes with an inner diameter of 2 cm with a stopper (no) c Method c» The method and calculation for determining abrasion are described above. from h o t sp

Comment

All media, with the exception of the Musotea collagen sponge, are not elastic after coating.

The sample must be cut very carefully. When cutting with scissors, there is a lot of coating material

GF) delaminates, since the layer itself is solid. Patch E (Pyzogr E Raispy did not connect with the coating material at all. When lightly shaken, the entire coating was covered like a "carpet". The difference between Musotea's collagen sponge and other carrier materials was very evident.

Elasticity of a moistened carrier with an applied coating 60 Method 1. Coating of various carriers

A suspension of TaspoSotr 5 was applied as a coating to the surface measuring 2x4.5cm of each carrier. The amount of suspension corresponded to the technical conditions of TaspoSotr (5.5 mg of fibrinogen/cm2). The samples were dried. ve 2. Have you prepared a sample with an area of approximately 5-7 cm? of each coated carrier. The exact initial area of the dry sample was measured.

3. The sample was moistened and placed on an elastic latex sheet fixed in a special device (for more details, see the "Method" section). A stretching pressure was then applied to the latex sheet. After three stretches, the area of the carrier at the highest stretch was measured.

Description of the method

Device/chemicals

Peristaltic pump (IKA RA-5E)

Buffer fluid under pressure (with outlet holes)

Manometer MOO (measurement limits 0-250bar) 70 Glass funnel (diameter of hole 1:30mm; hole 2:15mm)

Silicone tubes and clamps, latex gloves (Zetreg May), scalpel, ruler graduated in millimeters, scissors

Physiological solution

Method

The following equipment was hermetically connected to the three outlets of buffer fluid under pressure with silicone tubes: a) peristaltic pump b) manometer c) glass funnel (to hole 2).

From a latex glove, a double sheet with dimensions of approximately 8xhvsm was cut out. This sheet was hermetically fixed on a glass funnel (hole 1).

A piece approximately 5-7 cm in size was cut out of the coated carrier with a scalpel.

The area of the sample (initial area) was measured. The cover of the sample was moistened with physiological solution and placed on a latex sheet. Then he was manually held to the latex sheet for about 1 minute. with

With a peristaltic pump, the latex sheet is split by applying a pressure of approximately 7Ombar. This operation was repeated twice with the release of the latex sheet after each stretch. After the third stretching, the area (length and width) of the carrier was measured at the greatest stretching of the latex sheet.

Calculation: | | sh

The characteristic covariance is the area of the noses and the stretching of the wedges 7 and the seal of the sample

F

- зв M « - s Commentary and Elasticity of the hydrated Musotei collagen sponge (TaspoSotr) is one of the important characteristics of the product. Elasticity plays an important role in cavity operations and chest surgery. After bonding, the carrier must have the ability to, for example, expand and contract with the lungs or intestines. E(Vizogre did not show elasticity at all. It immediately lagged behind the coating. In the course of -i examination of M/ShovzropF 5relia! and OrgavzKip? with the applied coating had structural defects. Their Use of the carrier with the applied coating in endoscopic surgery

Method (c) 1. Applying a coating on different media 50 The surface of each media measuring 2x4 cm was coated with TaspoSotr 5 suspension. The amount of suspension corresponded to the technical conditions of TaspoSotr (5.5 mg of fibrinogen/cm). The samples were dried. sl 2. Manipulations with coated media samples for use in endoscopic surgery and loss of coating as a result of these manipulations are documented by digital photo equipment.

Description of the method 59 Equipment

GF) Epadodosk: an endoscopy tool intended for the use of TaspoSotr F in endoscopic surgery (see Fig. 7). Digital photo equipment. name) 59 65 Taroiatr MY Kiy Oxidized cellulose

Method

A series of photographs taken of each carrier: 9 1. Photograph: documentation of coated and uncoated carrier samples. 2. Photo: Specimens with the coating applied are inserted into the equipment for endoscopy (Epdodosk).

The sample must be manually smoothed and wound on the guide "finger". Then the sample is carefully inserted into a steel tube with a diameter of 1 mm.

Documentation of the sample, partially inserted into the Epaodosk tube. then 3. Snapshot: gently roll the sample. After that, it must be deployed. The coating separated from the carrier during this operation is collected from the side of the carrier.

The unfolded sample after being placed in the endoscope and the loss of coating as a result of this manipulation are documented.

Comment and nothing

TaspoSotri (horse collagen sponge with applied coating / Musoteia) in endoscopic surgery is the most responsible application of the product. The tape is placed in the equipment for endoscopy. The tube of this equipment usually has a diameter of 10-13 mm. Before placing in the tube, the tape is smoothed and then wound on the guide "finger", after which it is carefully inserted into the tube. Thus, the adhesion of the coating to and within the substrate must be strong, but the dry product must remain flexible enough to be bent and unfolded. The TaspoSotrB delivered to the operation site is carefully removed from the tube. Then it must be unfolded and placed on the surface of the wound. This often requires some manipulation to withstand which, the adhesion of the layer to the carrier must be strong enough.

Results The results are given in the attached Figures 1-6. The estimate of the loss of the coating material is given below: o

Orgovzkip? 30-4096

WE Povrop E ope 60-7096

WE ARE POVROP AND ZRESIA! 5OZo

EshivogF Raisp 60-7096 at 30 Taroiatr? MO Kpi 959 chI o

SoMPadep Zropde Musotei "596 Fr

Since E(PisogrF is a very hard support, the adhesion of the coating is very weak. Because of this, in the course of the "I experiment, EPisogr? lost almost all the coating. Compared to Musotea collagen sponge with a 3o coating applied, all other tested supports have a flat surface for application therefore, the coating lies on the carrier like a "flat carpet" and the elasticity of such dry carriers with the applied coating is quite low.

Often, when bending or unfolding, the coating itself is destroyed.

After the coated media were placed in the endoscope tube and then unfolded, all media, with the exception of the Musotea collagen sponge, lost a very significant amount of the coating, leaving large areas of C 70 uncoated. c The high elasticity of TaspoSotr in a dry or wet state is due to its structure and texture

From Musotea collagen sponge, which is a foamed material and has polygonal chambers inside. On the surface, these chambers are cut into cavities that increase the surface of the coating. When applying the coating, the suspension is evenly distributed on the structured surface. When drying, the solution containing fibrinogen and thrombin is fixed in 45 specified cavities as a solid substance. Thanks to this, TaspoSotr can be cut into pieces of the desired size and inserted into endoscopy equipment with little or no loss of coating material.

The high elasticity of TaspoSotrB in the dry state is a great advantage in comparison with all other coated carriers investigated. "iz" 20 Production of collagen sponge

The collagen sponge referred to in the text of this specification can be manufactured in the manner described below with reference to its general illustration in Figures 10 and 11.

It has been established that the successful coating of a collagen sponge with a fibrin glue preparation depends on the texture of the collagen sponge. Therefore, a method of making a collagen sponge with a defined texture is needed, in particular, in order for the collagen sponge to be suitable for coating with a fibrin glue preparation, and thus

GF) to receive material for healing and closing wounds. In addition, there is a need for a method of manufacturing a collagen sponge with improved physical characteristics compared to known sponges in terms of improved moisture content, elasticity, density and modulus of elasticity. There is also a need for a method of making a collagen sponge that is air- and moisture-tight in the sense that, when applied to a wound, it does not allow air or liquid 60 to be absorbed through the collagen sponge.

Based on the above, the method of manufacturing collagen sponge can consist of the following stages: - preparation of collagen gel; - adding air to collagen gel and mixing to obtain collagen foam; - drying of collagen foam to obtain a dry block of collagen sponge inside the chamber; bo - separation from the collagen sponge block of parts of the sponge with a chamber diameter greater than 0.75 mm and less than

4mm or cameras with a diameter of no more than Zmm on average.

In this context, the term "diameter of the chamber" should be understood as the greatest distance in a straight line between the walls of the chamber, that is, the greatest diagonal distance of the chamber in a straight line. Cameras can be polygonal, for example octagonal, in shape.

It has been established that the diameter of the chambers is greater than 0.75 mm and less than 4 mm or the diameter of the chambers on average does not exceed 3 mm, making the collagen sponge particularly suitable for covering it with fibrin glue. In a preferred embodiment, the collagen gel has a dry weight in the range of 2-20 mg per 1 g of gel, for example, 4-18, 5-13, 6-11 mg per 1 g of gel. The dynamic viscosity of the collagen gel is preferably equal to 2-20Ns/cm 2, for example, 4-10, 70. 8-VNs/cm2, The moisture content in the collagen sponge preferably does not exceed 2095, for example, 10-15, approximately 1895.

The modulus of elasticity of collagen is preferably in the range of 5-100N/cm 2, for example, 10-50N/cm?, and the density of the sponge is preferably equal to 1-10mg/cm3, for example, 2-mg/cm3.

It has been found that the collagen sponge made in the above manner is air- and moisture-tight in the sense that, when applied to a wound, it does not allow air or liquid to pass through the collagen sponge. 12 Liquids are absorbed by a sponge. This effect is achieved mainly due to the fact that the stage of adding air to the collagen gel and mixing allows to obtain a collagen sponge with a volumetric structure with chambers separated and completely surrounded by walls of collagen material, unlike known collagen sponges that have a fibrous structure .

The collagen gel may consist of material of various types, for example mammalian type !, I or I or derived from transgenic or recombinant sources, but all other types of collagen may be used.

The collagen may be derived from tendons selected from the group consisting of equine, human and bovine tendons.

The collagen gel may additionally or instead contain recombinant collagen material.

The collagen content in the separated parts of the sponge is preferably equal to 50-10095 in terms of the weight of the dry substance of the sponge, for example, 75-100, 80-100, 85-100, 90-100, 92-100, 92-98, 93-97, 94 -96905. with

The stage of preparation of collagen gel consists of the following stages: Ge) - preservation of tendons at a temperature from -10 to -302C and cleaning of tendons from shells; - removal of extraneous protein from tendons; - reduction of the content of pathogenic microorganisms in the tendons; - swelling of tendons; yuyu - homogenization of swollen tendons. "Her

The task of the stages of preservation, cleaning from shells, removal of protein, reduction of the content of pathogenic microorganisms and swelling is the purification of raw materials, and the stage of homogenization is to obtain collagen in the form of a gel. at

The stage of reducing the content of pathogenic microorganisms mainly includes the addition of acid to the tendons, "And for example, organic acid, for example, lactic acid. Next, an organic solvent is preferably added to the tendon, for example, alcohol, for example, ethyl alcohol. Next, the tendon swelling stage mainly includes adding lactic acid to the tendons. The lactic acid used can be 0.40-0.5095%, in particular, 0.4596%.

The step of swelling the tendons may include holding the tendons at a temperature of 4-252C, for example, at a temperature of 10-202C, for 48-200 hours, for example, for 100-200 hours. 7. The stage of homogenization of swollen tendons is preferably carried out in such a way as to obtain the size of collagen gel fragments, that is, fibrous balls, with a diameter of 0.8-0.12 cm, for example, approximately 1 cm. Next, the physical characteristics of the collagen gel are preferably as stated above.

The corresponding characteristics can be achieved, for example, by carrying out the homogenization step of the swollen tendons in a toothed disc mill or other suitable homogenization equipment. - 15 The stage of adding air to the collagen gel and mixing preferably includes the following stages: - adding ambient air to the gel and mixing with a mixer to obtain foam and collagen; o - feeding the foam of the mixed gel into the channel for fractionation; - separation of collagen gel and collagen foam located in the channel for fractionation. At least some of the collagen gel separated from the collagen foam in the fractionation channel can be returned to the mixer. In this case, the ratio of the amount of collagen gel returned to the mixer from the channel for fractionation to the amount of fresh gel fed into the mixer is preferably equal to 0.1-0.5. The stage of separation of collagen gel and collagen foam preferably includes the following operations: - separation of the selected part of the collagen foam located in the channel for fractionation; 59 - supply of the selected part of the collagen foam from the fractionation channel for drying.

GF) In the preferred embodiment of this method, the temperature in the channel for fractionation is maintained at

HF ranges from 15 to 402С, for example, 20-2596.

After mixing the collagen gel with air, the collagen foam can be homogenized within 2-4 minutes. Before the operation of drying the collagen foam and after mixing the collagen gel with air in the collagen foam, a neutralizing agent can be added to bring the pH value from the usual 2.5-3.5 to the pH value in the collagen foam of 6.5-8.5. An ammonia solution can be used as a neutralizing agent, and the collagen foam is preferably neutralized within 5-30 hours, for example, 10-20 hours, for example, approximately 24 hours.

Before drying the collagen foam, it is preferably poured into a drying container in such a way that no air gets into the foam when it is poured.

Drying is preferably carried out at a temperature between 15 and 60 °C, for example, between 20 and 4090,

for 50-200 hours, for example, 100-150 hours, to obtain a dry collagen sponge. Drying can be carried out at a pressure slightly less than atmospheric, for example at a pressure between 700 and 9bObar, for example approximately 800bar.

A collagen sponge made in the above-described manner should preferably meet at least one of the following criteria: - pH: 5.0-6.0; - lactic acid content: no more than 5905; - ammonium content: no more than 0.595; 70 - soluble protein content, calculated as albumin content: no more than 0.595; - content of ash sulfates: no more than 1.095; content of heavy metals: no more than 20 million"; - microbiological purity: no more than 1 OZKUO/G; - collagen content: 75-10090; - density: 1-10mg/smu, for example, 2-7mg/cm; - modulus of elasticity: 5- 100N/cm, for example, 10-BON/cm3.,

The operation of separating parts of the collagen sponge can be a division of the collagen sponge into several parts by cutting. The resulting parts can be given any desired shape, for example, conical, cylindrical, including cylindrical with an annular cross-section, rectangular, polygonal, cube-shaped, and flat sheets, or they can be converted into pellets by a suitable granulation method.

As follows from the above, collagen sponge can be produced in a way that includes the following stages: - preparation of collagen gel; - adding air to the collagen gel and mixing to obtain collagen foam; - drying of collagen foam to obtain a dry block of collagen sponge contained inside the chamber; - separation from the collagen sponge block of parts of the sponge with the following properties: c - modulus of elasticity: 5-100N/cm?; Ge) - density: 1-10mg/cm3; - diameter of cameras: greater than 0.75 mm and less than 4 mm or diameter of cameras on average no more than Zmm.

IS)

Claims (68)

30 Formula of the invention " 1. A suspension containing fibrinogen, thrombin and alcohol, which differs in that it is obtained in a way that includes: "I - preparation of a mixture of fibrinogen and alcohol; 35 - preparation of a mixture of thrombin and alcohol; - - mixing of a mixture of fibrinogen and a mixture of thrombin to obtain the specified suspension, which contains particles of fibrinogen and thrombin with an average particle diameter according to Roik U/aga in the range of 25-100 μm. " 2. The suspension according to claim 1, which differs in that the average diameter of the particles according to Roik Uuaga is within 70 ЗB-VOМКМ. N- c C. The suspension according to claim 1 or 2, which is characterized by the fact that its viscosity is such that only 90-120 ml of the suspension under the influence of gravity flow out through the lower opening of the container, which has a cylindrical part with an inner diameter of 40-5 Ω and an internal length of 55-65 mm and a conical lower part of a height of 17-23 mm with a lower round hole at the lower end of the said conical part with a diameter of 2 mm, in 25-75 seconds. 45 4. The suspension according to item C, which is characterized by the fact that the specified volume of the suspension flows out through the lower hole in 7 30-50 seconds. h 5. Suspension according to any one of claims 1-4, which differs in that it also contains aprotinin. 6. The method of preparing a suspension of fibrinogen and thrombin, which differs in that it includes the following stages: o - prepare a mixture of fibrinogen and alcohol; part 20 - prepare a mixture of thrombin and alcohol; - mix a mixture of fibrinogen and a mixture of thrombin sl to obtain the indicated suspension, which contains particles of fibrinogen and thrombin with an average particle diameter according to Roik U/aga in the range of 25-100 μm. 7. The method according to claim 6, which differs in that at the stage of preparation of the fibrinogen mixture, small particles of fibrinogen are pre-selected in order to obtain particles with an average diameter according to Roik Uuaga in the range of 25-100 μm. GF) 8. The method according to item b or 7, which differs in that previously selected small particles of fibrinogen are mixed in alcohol to obtain a mixture of fibrinogen. 9. The method according to any of claims 6-8, which differs in that at the stage of preparing the mixture, it is homogenized. 10. The method according to claim 9, which differs in that the specified mixture is homogenized at a temperature ranging from 0 60 to 12C. 11. The method according to claim 10, which differs in that the mixture is homogenized at a temperature ranging from 2 to 820. 12. The method according to claim 11, which differs in that the temperature is lowered during homogenization. 13. The method according to any one of claims 6-12, characterized in that the thrombin contains at least one of the following thrombins: human thrombin, bovine thrombin and recombinant thrombin. for 14. The method according to any one of claims 6-13, which is characterized in that the thrombin contains at least one of the following thrombins: human thrombin and recombinant thrombin. 15. The method according to any of claims 6-14, which is characterized by the fact that the suspension also contains aprotinin. 16. The method according to any of claims 6-15, which differs in that ethyl alcohol is used. 17. The method according to claim 16, which is characterized by the fact that ethyl alcohol is anhydrous. 18. The method according to any of claims 6-17, which is characterized by the fact that the stage of mixing the mixture of fibrinogen and the mixture of thrombin is carried out while stirring the suspension. 19. The method according to claim 18, which differs in that the mixing is carried out at a temperature in the range from 0 to 1296. 70 20. The method according to claim 19, which is characterized in that the mixing is carried out at a temperature in the range from 2 to 826. 21. The method of coating the carrier from a suspension containing fibrinogen and thrombin, which is characterized by the fact that the suspension is made in a way that includes: - preparation of a mixture of fibrinogen and alcohol; - preparation of a mixture of thrombin and alcohol; - mixing of a mixture of fibrinogen and a mixture of thrombin to obtain the specified suspension, which contains particles of fibrinogen and thrombin with an average diameter of particles according to Roik UUaga in the range of 25-100 μm, and the specified method of coating includes: - delivery of a suspension of fibrinogen, thrombin and alcohol to a place next to carrier; - applying the suspension to the surface of the carrier to be covered. 22. The method according to claim 21, which is characterized by the fact that the carrier is collagen. 23. The method according to claim 22, which differs in that the collagen carrier is a collagen sponge. 24. The method according to claim 23, which is characterized by the fact that the collagen sponge meets at least one of the following SM criteria: o - pH value: 5.0-6.0; - lactic acid content: no more than 5905; - ammonium content: no more than 0.595; - soluble protein content, calculated as albumin content: no more than 0.595; Io) - the content of sulfate salts: no more than 1.095; "- content of heavy metals: no more than 20 million"; - microbiological purity: no more than 1 OZKUO/G; o - collagen content: 75-10090; "E - density: 1-10mg/cm3; - modulus of elasticity: 5-100N/cm7 - 25. The method according to claim 23 or 24, which is characterized by the fact that the carrier is a collagen sponge, which is made in a way that includes: - preparation of a collagen gel; « и - addition of air to the collagen gel and mixing to obtain collagen foam; - o - drying of collagen foam to obtain a dry block of collagen sponge contained inside the chamber; c - separation from the collagen sponge block of sponge parts with a chamber diameter greater than 0.75 mm and less than 4 mm, or with chambers with an average diagonal size of Zmm. 26. The method according to any of claims 21-25, which is characterized by the fact that the suspension is applied to the carrier at an ambient temperature in the range from 0 to 12960. -1 but 27. The method according to claim 26, which differs in that the suspension is applied to the carrier at an ambient temperature in the range from 1 to 10290. - 28. The method according to claim 27, which differs in that the suspension is applied to the carrier at an ambient temperature in the range from 2 to 820. 29. The method according to any one of claims 21-28, which is characterized by the fact that the suspension is applied to the carrier at a relative humidity of the surrounding atmosphere in the range of 75-9996. with 30. The method according to claim 29, which differs in that the suspension is applied to the carrier at a relative humidity of the surrounding atmosphere in the range of 85-95905. 31. The method according to any of claims 21-30, which differs in that by one cm? 0.08-0.12 ml of suspension is applied to the surface of the carrier to be covered. 32. The method according to any of claims 21-31, which is characterized by the fact that the suspension is evenly distributed over the given iF) width of the surface to be covered, so that the mass of fibrinogen per unit surface area does not exceed 2590. 33. The method according to any of claims 21-32, which is characterized by the fact that an applicator having at least one nozzle is used to apply the suspension to the carrier, and the pressurized jet applicator has the ability to supply the suspension through the nozzle when the carrier is moved and nozzles relative to each other. 34. The method according to any one of claims 21-32, characterized in that for applying the suspension to the carrier, an applicator with a container having several separate outlet openings is used, in which the suspension from the container is fed under pressure through the outlet openings to the carrier. 65 35. The method according to claim 34, which differs in that when the suspension is applied to the carrier, the latter and the applicator move relative to each other in the direction of transportation. 36. The method according to claim 35, which differs in that the speed of movement is in the range of 0.025-0.05 m/s. 37. The method according to claim 36, which differs in that the speed of movement is in the range of 0.03-0.04 m/s. 38. The method according to any of claims 34-37, which is characterized by the fact that the flow rate of the suspension applied through the applicator to the carrier is in the range of 400-6b0Oml/min. 39. The method according to claim 38, which differs in that the consumption of the suspension is in the range of 470-55O0ml/min. 40. The method according to claim 39, which differs in that the consumption of the suspension is in the range of 495-505 ml/min. 41. A method of drying a suspension of fibrinogen, thrombin and alcohol, applied as a wet coating on the surface of the carrier to be covered, and the specified suspension contains particles of fibrinogen and thrombin with an average diameter of 70 particles according to Roik Uuaga in the range of 25-100 μm, and the specified method includes an exposure stage on the carrier with the applied coating, the pressure is below 100O0mbar so that the dried surface of the coated carrier and the fixation of the dried coating on this surface are obtained. 42. The method according to claim 41, which differs in that the suspension is obtained in the following way: - preparation of a mixture of fibrinogen and alcohol; - preparation of a mixture of thrombin and alcohol; - mixing of a mixture of fibrinogen and a mixture of thrombin to obtain the specified suspension, and as a carrier, a collagen sponge is used, made in a way that includes the following stages: - preparation of a collagen gel; - adding air to the collagen gel and mixing to obtain collagen foam; - drying of collagen foam to obtain a dry block of collagen sponge contained inside the chamber; - separation from the collagen sponge block of parts of the sponge with a chamber diameter greater than 0.75 mm and less than 4 mm, or with a chamber with an average diagonal size of Zmm, and a coating is applied to the collagen sponge by: - delivery of a suspension of fibrinogen, thrombin and alcohol to a place nearby with collagen sponge; sch - application of the indicated suspension on the covered surface of the collagen sponge. 43. The method according to claim 41 or 42, which is characterized by the fact that the carrier with the applied coating is dried under the specified (8) pressure at a temperature ranging from 0 to 1296. 44. The method according to claim 43, which is characterized by the fact that the carrier with the applied coating is dried at the indicated pressure at a temperature in the range from 1 to 1026 °C 45. The method according to claim 44, which is characterized by the fact that the carrier with the applied coating is dried under the specified pressure at a temperature in the range from 2 to 826. With 46. The method according to any of claims 41-45, which is characterized by the fact that the carrier with the applied coating is dried under the specified pressure at a relative humidity of the surrounding atmosphere in the range of 75-99965. 47. The method according to any one of claims 41-46, which is characterized by the fact that the carrier with the applied coating is dried under the indicated pressure at a relative humidity of the surrounding atmosphere in the range of 85-9595. - 48. The method according to any one of claims 41-47, which is characterized by the fact that during drying, the carrier with the applied coating is blown with a stream of air. 49. The method according to any of claims 43-48, which is characterized by the fact that the carrier with the applied coating is kept at the specified temperature for at least 1 hour. 50. The method according to any of claims 43-48, which is characterized by the fact that the carrier with the applied coating is kept at the indicated temperature for at least 2 hours. and 51. The method according to any one of claims 43-48, which is characterized by the fact that the carrier with the applied coating is kept at the specified temperature for at least 4 hours. 52. The method according to any one of claims 41-51, which is characterized by the fact that the area of the dried surface to be covered is not less than 7595 of the area of this surface in the wet state. -and 53. The method according to any of claims 41-51, which is characterized by the fact that the area of the dried surface to be covered, 1" is at least 8095 of the area of this surface in the wet state. 54. The method according to any one of claims 41-53, which is characterized by the fact that the moisture content of the carrier and the dried coated (a) surface does not exceed 1290 by weight. drive 50 55. The method according to any one of claims 41-53, which is characterized in that the moisture content of the carrier and the dried coated surface does not exceed 895 by weight. sl 56. The method according to any one of claims 41-55, which is characterized by the fact that said suspension also contains aprotinin. 57. A collagen sponge coated with fibrinogen and thrombin, which is characterized by the fact that the coated collagen sponge is obtained by a method that includes the following stages: - production of a collagen sponge by a method that includes: - preparation of a collagen gel; o - adding air to the collagen gel and mixing to obtain collagen foam; co - drying of collagen foam to obtain a dry block of collagen sponge containing inside the chamber; - separation from the collagen sponge block of sponge parts with a chamber diameter greater than 0.75 mm and less than 60 4 mm, or with a chamber with an average diagonal size of Zmm; - application of a suspension of fibrinogen, thrombin and alcohol on the covered surface of the collagen sponge, and this suspension contains particles of fibrinogen and thrombin with an average particle diameter according to Roik Umaga in the range of 25-10 Ωm, and - application to the carrier with the applied coating pressure below 1000 mbar yes , providing a dried surface 65 of the coated carrier and fixing the dried coating to the coated surface, wherein the coated collagen sponge has at least one of the following properties: - the suspension is evenly distributed over the given width of the surface to be covered, so that the mass of fibrinogen per unit area of the surface to be covered does not exceed 25,905; - when shaking a sample of the coated material measuring 1 x 5 cm in a test tube in a Mirgoii shaking machine at a rotation frequency of approximately 800-1200 rpm. within 2 minutes, the abrasion of the coating is less than 2.Omg/cm?. 58. A coated collagen sponge according to claim 57, characterized in that the suspension has a moisture content of 20-80 mg/ml. 59. A coated collagen sponge according to claim 57, characterized in that the suspension has a moisture content of 70 24-32mg/ml. 60. A coated collagen sponge according to any of claims 57-59, which is characterized in that the thrombin content in the suspension is equal to 20-DA0OMO/ml. 61. A coated collagen sponge according to any of claims 57-59, which is characterized in that the thrombin content in the suspension is 24-33 IU/ml. 62. A coated collagen sponge according to any one of claims 57-61, which is characterized by the fact that the thrombin content in the coated surface is on average in the range of 2-4 AMO/cm2, 63. The coated collagen sponge according to any of claims 57-61, which is characterized by the fact that the thrombin content in the coated surface is on average in the range of 2.3-3.3 IU/cm7. 64. A coated collagen sponge according to any one of claims 57-63, characterized in that the thrombin content at any site on the coated surface does not exceed 5 BMO/cm2. 65. A coated collagen sponge according to any one of claims 57-63, characterized in that the thrombin content at any site on the coated surface does not exceed 3.8 IU/cm. 66. A coated collagen sponge according to any of claims 57-65, which is characterized by the fact that the microbiological purity of the carrier with the applied coating is no more than 4 ACUO/cm7, s 67. A coated collagen sponge according to any of claims 57-65, which is characterized by the fact that (o) the microbiological purity of the coated carrier is no more than 2.25 CFU/cm, 68. The use of a collagen sponge with an applied coating for gluing and closing tissues and hemostasis, and the collagen sponge has a coating of fibrinogen and thrombin, which is characterized by the fact that the collagen sponge with an applied coating is obtained in a way that includes the following stages: - production of collagen sponge sponges in a way that includes: "- preparation of collagen gel; o - adding air to the collagen gel and mixing to obtain collagen foam; - drying of collagen foam to obtain a dry block of collagen sponge contained inside the chamber; "- separation from the collagen sponge block of sponge parts with a chamber diameter greater than 0.75 mm and less than 4 mm, or with a chamber with an average diagonal size of Zmm; - application of a suspension of fibrinogen, thrombin and alcohol on the covered surface of the collagen sponge, and the said suspension contains particles of fibrinogen and thrombin with an average particle diameter according to Roik Umaga in the range of 25-10 Ωm, and « 20 - application to a carrier with a pressure coating applied below 100Ombar so that a dried surface is obtained (Zh c carrier to be covered and fixation of the dried coating on the surface to be covered, and the collagen sponge with the applied coating has at least one of the following properties: c - the suspension is evenly distributed over the given width of the surface to be covered , so that the mass of fibrinogen per unit area of the covered surface does not exceed 25905; - when shaking a sample of coated material measuring 1 x 5 cm in a test tube in the apparatus - and for shaking Mibgoi at a rotation frequency of approximately 800-1200 rpm during 2 minutes of abrasion of the coating is less than 2.Omg/cm?