JPS61178927A - Dry medicine containing fibrinogen and manufacture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a dry fibrinogen-containing preparation having a foam-like or wool-like structure obtained by freeze-drying. Such dry formulations are completely absorbed and can be used, in particular, as a support for other active ingredients useful in therapeutic processes.

Furthermore, the present invention also relates to a process for producing such dry formulations.

Prior Art A dry preparation of this type is described in German published patent application No. 30375.
No. 13, this application relates to a colladan wound dressing obtained by freeze-drying a solution containing both collaine and fibrinogen. The resulting dry material may additionally contain any pharmaceutically active ingredients such as antibiotics. However, as discussed below, the significant amounts of colladan contained in wound dressings may be harmful, especially if it is absorbed.

Furthermore, freeze-dried tissue adhesives (for example, German Published Patent Application No. 3002934) are known, which mainly consist of fibrinogen and albumin in addition to fiber degradation inhibitor and factor (2), as well as glycan and glucose. and may additionally contain heparin.

However, this tissue adhesive is not used in the form of a dry preparation on the wound; water is added to reconstitute it into a concentrated fibrinogen solution, and then the solution is mixed with thrombin and cac4'.
4. Use in addition.

Furthermore, from German Published Patent Application No. 2852319 an absorbable hemostatic material for use in controlling bone bleeding is known, which is a bath-compatible, biocompatible material.
It contains a hemostatic powder, which is a mixture of fibrin and colladan powder, in a tl-rich matrix.

Finally, a molded article is known from USP 3,523,807 which is completely resorbed after insertion and is mainly free of fibrin. To produce this finished product, calcium chloride is mixed with plasma, water is added to make a paste, the paste is placed in a mold and pressurized to obtain the desired molded product. This is then placed in a bath containing formaldehyde and cured.

Freeze-drying of aqueous protein solutions is known to those skilled in the art and results in a fluffy, soft, foamy, felt-like or wool-like structure with numerous cavities; They result in dry formulations with a high absorption capacity for body fluids.

It is also stated, for example, in German Published Patent Application No. 2,625,289 that preparations having a schifert-like or wool-like fiber structure can be obtained by freeze-drying an aqueous solution of colladan. Such dry collagen preparations are characterized by high mechanical strength, which allows the collagen wool to be cut and bent.

However, when a dry preparation is made by freeze-drying the Fipurinogun water bath solution under similar conditions, the foamed groove-a is formed. t) The preparation is obtained, but it breaks easily and becomes crumbly. Due to their insufficient mechanical strength, such formulations may be unsuitable as carriers for pharmaceutically active ingredients.

Hemostatic effect and high stability (mechanical strength, storage stability)
Despite this, collagen loadings prepared by freeze-drying or the like have not been completely satisfactory when used as absorbable inserts. Disadvantages include, for example, a long residence time of more than 6 weeks at the wound site, and undesirable collagen shadows during the healing process, such as in the case of fractures. #Temples can be mentioned.

Often known collagen preparations can exhibit denaturation during separation from their natural raw materials and formulation, turning them into insoluble preparations with poor absorption performance. In contrast, readily atrial collagen preparations always contain pH-acidifying salts, which, when introduced into a wound, create an acidic environment there and neutralize it. Doing so is an unnecessary burden on the tissue and impedes the healing process. Optimal development of wound healing and fibrin formation by induction of thrombin from insoluble fibrinogen requires an environment with a physiological pH value of approximately 7.36.

As a final note, collagen is always present in physiologically available form in endogenous structures, tissues, and body fluids at the wound site, and it is necessary to replenish significant amounts of additional collagen. It's neither good nor desirable.

Problems to be Solved by the Invention In view of the above points, the object of the present invention is to provide the above-mentioned types of collagen preparations which have the advantages of conventional collagen preparations despite not containing any or substantially no collagen. The purpose of the present invention is to produce a dry preparation containing fipurinogun. This formulation is more similar to natural wound closure agents than previous ones in terms of its composition, physiological properties and absorption behavior, and can be administered directly without supplementary procedures such as the addition of additional active ingredients. It can be applied to a wound and can enhance the hemostasis effect on the wound.

According to the invention, this object is achieved by a wool-like material obtained by freeze-drying. This woolly material contains at least a catalytically effective amount of thrombin plus about 10 to 9
5-layer minoku - cent fibrin and about 5-9 czt! It is essentially composed of Purcet (7') fibrinodan.

By the method of the present invention, the dry preparation'f: K produced
The entire fibrin is made in situ in a water bath containing fipurinogun and thrombin, and the resulting reaction mixture is then lyophilized.

Another aspect of the present invention is that such dry formulations can be used extensively in a variety of applications as a support material and as well as a reservoir for active ingredients to assist in the therapeutic process. . The amount and duration of release of the active ingredient from this dry formulation depends on the fibrin content.
It can be controlled by additional components and by predetermining the degree of crosslinking and/or polymerization of the fibrin.

Preferred application examples and other embodiments of the present invention are disclosed in the following claims (2).

The present invention was made based on the following observations. That is, the dry formulation obtained by freeze-drying a reaction mixture containing in situ generated fibrin in addition to fibrinogen and at least a catalytic amount of thrombin is surprisingly stable when compared to the stability of the freeze-dried collagen formulation. It shows high stability. Even without the addition of other substances and factors that aid in the coagulation of blood, such dry preparations exhibit a very high hemostatic effect, especially with the added action of active thrombin and fibrinogen.

The fibrin, which is preferably produced in situ and used in the form of a dry formulation, is particularly duplexed, solid fibrin, has a high biological activity, is cross-linked only in the longitudinal direction, i.e., only through its γ-links, and is is used as a starting point for more intense fibrin production at the wound site.

To prepare the dry formulations of the invention, one starts from an aqueous solution or an aqueous solution consisting predominantly of water. This initial solution may be water,
It may be human serum or an aqueous salt solution. The salt bath solution may be physiological saline or physiological saline fortified with Caα and phosphate. The pll and salt content of this initial solution shall correspond primarily to physiological conditions.

Depending on the situation, it may be appropriate to use an aqueous solution consisting mostly of water, containing a water-soluble organic solvent in addition to water as the main ingredient, for example to increase the solubility of certain active pharmaceutical ingredients.

Suitable organic solvents include monohydric alcohols such as ethanol, ink-free lobanol, polyhydric alcohols such as glycerin, polyglycols, cyclic ethers such as dioxane, and the like. The maximum content of organic solvent shall be selected such that it does not denature and/or reduce the activity of the enzymes used and does not inhibit the drying of the frozen material. Generally, the proportion of organic solvent should not exceed 20 cents by volume of the total mixture.

Add human fibrinogen to the first solution.

Suitable fibrinogen preparations are commercially available.

For example, it can be purchased from Basing Perke in Malpurg. Furthermore, suitable fibrinogen formulations can be made according to the following Gluceth.

Human plasma was cooled to 4°C and β-alanine (2 molar solution in ethanol) was added under stirring, followed by further addition of ethanol.
- addition to precipitate crude fibrinogen. This crude fibrinogen was centrifuged and Tris buffer solution (
Dissolve in 0.01M solution (pH 7.4), add 2M glycine and precipitate again. The separated precipitate is dissolved in 0.9% NAα aqueous solution, dialyzed against the same solvent to remove salts and then lyophilized.

The microcrystalline fibrinogen obtained in this way has a molecular size of 340.000±5%, is partially digested and degraded by the α-chain, and is then introduced into body fluids. and immediately start polymerization. The proportion of coagulable fibrinogen in solution amounts to at least 85%.

Add about 10 to 90 η of fibrinogen per IWL of solution into the above initial solution, preferably kept at room temperature. Preferably, the fibrinogen concentration t
A relatively high concentration of -50 to 80 Da/d is used. The formulation obtained by lyophilization has the same volume as the original solution, unless foaming is observed in the solution. Therefore, increasing the fibrinogen concentration in the initial solution will result in a higher concentration and mechanical strength in the final product.

After addition, the fibrinogen is dissolved in the initial solution; stirring may be carried out at room temperature for this purpose.

Another method does not require first separating the fibrinogen into a pure solid form and then adding it to the water bath solution. For example, the precipitate described above in connection with the separation of fibrinogen, obtained after precipitation with glycine, can be dissolved in a 0.9% Na11J aqueous solution to adjust the desired concentration. Additional additives can then be added directly to this solution, as described below. As another fibrinogen solution, a fibrinogen solution obtained by separating crude fibrinogen as a cold precipitate from residual serum containing serum proteins and various factors can also be suitably used.

This is, for example, the process of German Published Patent Application No. 3002934. In this regard, it is important to isolate as thoroughly as possible the enzymes and/or factors that trigger spontaneous fibrin formation, so that the initial solution contains significant amounts of citrate, phosphate, etc. Stabilizers like salt and oxalate 2! -No need to add.

The fibrinogen solutions produced by these processes can be sterilized, particularly to inactivate viruses such as liver moxibustion virus.

For this purpose, it is also possible, for example, to treat with β-guggulolagtone and then to apply UV irradiation. High energy r- or x-ray irradiation is also possible.

Selected additives and active ingredients can then be added to the fibrinogen solution.

The flexibility, mechanical strength and stability of this dry formulation can be adjusted by incorporating various glycoproteins. For this purpose, albumin, sud? Protein, fipronectic: y (
fibronectin) and/or globulin (α
-1β-r-globulin) can be added to the fibrinogen solution. The addition of these glycoproteins is about 3 to 40 weight percent, preferably about 5 to 25 weight percent, based on the finished dry formulation. These glycoproteins also function as desiccants and stabilizers, preventing the activity of coagulation enzymes from decreasing during long-term storage.

If this dry formulation is used in particular as a support or storage material for an active ingredient, it can be added to the fibrinogen solution, provided that the active ingredient is water-soluble and is not impaired by freeze-drying.

In this case, [active ingredient J (active 5ubst
The term ance is used in a very broad sense and refers to any tissue that is used parenterally for the cure, mitigation, treatment and/or prevention of disease in humans or animals, or anything that affects the functioning of an organism. It shall contain a composition of.

In this regard, substances that exhibit antibacterial activity are primarily considered, especially antibiotics. Suitable antibiotics include, for example:

Aminoglycoside antibiotics group such as gentamicin, nogopiocin (no.
Lactone antibiotics such as vobiocin), baycillin or 7% $-/cillin (
penicillin groups such as amox-icillin), team7 x 2: l-/l/ (tiamphe
chloramphenicol and its derivatives such as nicol), stref')mycin, tetracycline, and the like.

In addition, other substances exhibiting antibacterial effects include, for example, sulfonamides. Aminoglycoside antibiotics, sometimes gentamicin, are particularly preferred in this case due to their broad spectrum of action. Furthermore, combinations of different antibiotics can also be used, for example a combination of gentamicin and tetracycline. Active ingredients with other indications that are suitable for this purpose include, for example, antibacterial agents such as salicylic acid or undecenoic acid, pyranone
anti-inflammatory agents such as razolones, and anti-cellular agents such as prednisone.

The amount of these active ingredients added varies within a wide range and depends primarily on the activity of the active ingredients. Based on the total weight of the dry product, the amount of these active ingredients is approximately 0.1 to 1
0 weight percent, preferably about 2 to 6 weight percent. For example, a solution containing 500 to 10,000 units of gentamicin per 11 liters of fibrinogen solution has proven particularly suitable.

In conjunction with the above-mentioned active ingredients, additives can also be added which control the time and amount of release of the active ingredients. Effective additives of this type include collagen and crosslinked fibrin. The effectiveness of these additives depends on their compatibility with the selected active ingredients. In the case of many active ingredients, especially in the case of the important antibiotic group, gentamicin, the amount of additive that controls the total release of the active ingredient is approximately 2-12% of the total weight of the dry formulation.
A weight percent, preferably 4 to 10 weight percent, has proven sufficient. Colladan amounts of about 0.5 to 1 weight percent are particularly preferred for this purpose. For this purpose, it is sufficient to add an appropriate amount of aqueous solution of collagen whole fipurinodan solution, or to dissolve Hafipurinoke 07 in the corresponding collagen water bath solution. mosquito\
This amount of collagen is easily dissolved in the overall formulation, so that the aforementioned problems (acidic pn and/or non-physiological salt concentrations) do not occur. Furthermore, the amount of active ingredient released is influenced by the amount of fibrin and/or the degree of crosslinking of the fibrino. In most cases, the greater the degree of crosslinking of lefibrin with a high fibrin content, the slower the release of the active ingredient will be.

A suitable degree of fibrino crosslinking can be adjusted, for example, by adding gold glutaraldehyde.

In addition, fibrinolysis inhibitory factor (fibrinolysis inhibitor)
nhibiter) f can be added. It is preferred to add a relatively large amount of fibrinolytic inhibitor. That is, at least 5. O00 units (so-called kallikrine inactivator units), preferably 910.000 units or more of fibrinolytic inhibitor per ml of solution are added. Fibrinolytic inhibitors that come into consideration include, for example, grasminodan activator inhibitor or one or more of the antigrasmins, such as alpha-1 antiplasmin, alpha-macroglobulin or aprotinin, and also 8-aminocabu O7 acid. (a-aminocaproic acid
) and/or trypsin inhibitors. Reno 9
- Bayer AG of Riesen has announced the launch of “Trasilol” (“Tr
It is known to be suitable for the natural fibrinolytic inhibitor fi% sold under the trade name ``asylol'').

The above active ingredients are selected according to the intended use, and one or more of the other active ingredients are added to the fibrinodan bath solution, completely or substantially dissolved, and then thrombin is added. As you know, thrombin
n) are fibrinopeptides A and B (fibrinopeptides A,
B) f can be separated. At this time, fibrino monomer is obtained. This reacts spontaneously with other fibrin monomers to form a final polymer of 1/lj fibrin molecules. The added thrombin is at least 910 O per η under known standardized conditions.
00 units (American National Institute)
It shall have a biological activity value of NIH unit according to the standards of Teof Health. Suitable formulations are commercially available. For example, from the Hofmann-Rotsch company in Grensach, Baden, r) Vostasin J (“TOI”) O8.
Suitable thrombin preparations in the form of crystalline crystals with biological activity values of at least 3.000 units per rng of preparation are sold under the trade name "tas in").
In addition to thrombin, this formulation contains known stabilizing agents and supporting materials. )mlL Some of the activity of thrombin can also be added by the form of precursors that form thrombin, such as Bronton. Concentrates of prothrombin are also commercially available. For example, Vienna Neumuno (I)
rrmuno AG) PP5B formulation. Furthermore,
Thrombin and prothrombin-containing retrofit preparations can be separated from commercially available prothrombin conjugates by column chromatography, or extracted from human plasma using barium sulfate and separated from the resulting crystal precipitate. .

The amount of active thrombin added is determined by various factors. In terms of process conditions, high thrombin levels accelerate fibrino formation. So thrombin's ll! If the temperature is high, the reaction mixture must be deep cooled after a relatively short time so that the amount of fipurinodan is still sufficient. For the final product and its various uses, a relatively high concentration of thrombin is desirable in wound dressings because it accelerates hemostasis and at the same time provides additional fibrinogen.

For applications such as supporting materials for active ingredients such as antibiotics,
The t of thrombin in the final product is not very important. In this case, it is sufficient to add fine thrombin, which can be supplied within a reasonable time to produce the desired large amount of fibrino, which is more than 50% of the figurinodan.
This is enough to convert it into 7 f fibrin. In such cases, a catalytically active amount of thrombin of at least 0.1 units, preferably about 5 to 10 units per ml of fibrinogen solution will be sufficient.

Unless unphysiologically high concentrations of salts and/or stabilizers are present, thrombin produces fibrin monomers that polymerize in water from fibrinogen. The properties of the tellimer from the monomer thus obtained vary depending on various factors. If the formulation containing fibrinogen and thrombin is left alone, all the fibrinogen will react within about 4-6 hours and a firm glue will be obtained. This P
When kVi is shaken, it breaks and becomes fibrin filaments. In this case, the α-
and is done via the Ur chain. Lyophilization of the resulting precipitate gives a hard and brittle product, which is not very suitable as a wound dressing.

This is because mechanical strength and flexibility are low, solubility is low, and decomposition is slow.

When fibrin monomer is polymerized alone, a soluble polymer with poor stability is obtained, but this is stabilized mainly by hydrogen cross-linking, electrostatic interaction, water utilization reactions, etc. Covalent bonds (coval) between adjacent fibrin molecules
ent bond) t-formation increases stability. As is known, 1 - this requires the presence of a factor (2), which can also be activated by thrombin in the presence of Cactt, either in solid or dissolved form, depending on the formulation conditions. Fibrinogen practically always contains relatively small amounts or relatively large amounts of factor XIIIt. Under conditions for separating crude fibrinogen from plasma, factors
is separated along with crude fibrinogen.

The generation of covalent bonding states also takes place in the various side chains of adjacent fibrin molecules, which contribute to varying degrees of longitudinal and transverse fibrin polymer crosslinking.

As has been accomplished within the scope of the present invention, timely interruption of covalent bond formation can result in a soluble fibrin polymer that is primarily longitudinally crosslinked.

This is because the covalent bonds required for cross-linking in the lateral direction proceed at a slower rate than the covalent bonds required for cross-linking in the longitudinal direction. The onset of lateral crosslinking can be detected by a significant increase in the viscosity of the product. The viscosity of the fibrinogen/fibrin monomer solution was monitored to control the end point of γ-chain diquantization.

If the reaction is not interrupted at the correct time, the viscosity due to the action of σ-1 chain polymerization can reach up to 10 times the viscosity of the initial solution.

Fibrinogen/fibrinogen suitable for producing dry preparations according to the invention
The viscosity of the fibrin-septumer bath solution is twice that of the initial solution. This is because, at this time, the formation of doublets of the γ-chain is substantially completed, and the polymerization of the σ-chain has not yet progressed in earnest.

Depending on the concentration of thrombin and factor
It's a minute.

In many applications, particularly wound dressings, polymers of fibrin that are primarily longitudinally crosslinked are desired. This fibrin polymer is easily soluble, highly physiologically active, contains large amounts of thrombin in bound form, and is therefore available for rapid clotting of blood. Such fibrin is cross-linked primarily in the longitudinal direction, and consists mainly of γ-linked monodimeric fipilin. This is foamed or crosslinked in solution by adding glutaraldehyde if necessary.

As a basis for determining the extent of longitudinal and lateral fibrin polymer crosslinking, the formation of α-polymer and r-dimer can be measured.

For this purpose, sodium lauryl sulfate (sodi u
Dissolve the fibrin mixture in a buffer solution containing nlauryl 5 ulphate, add mercaptoethanol, and add mercaptoethanol. Separation is carried out electrophoretically in a lyacrylamide barrel.

If a primarily longitudinally cross-linked fibrin polymer is desired, the reaction time after thrombin addition can be up to 40 min.
shall be limited to minutes.

Preferably, about 10 to 30 minutes after adding thrombin.
minutes reaction time shall be given.

Under such conditions, the resulting fibrin 4-limer contains primarily gamma-linked dimers and is substantially free of sigma-linked polymers. Additionally, fibrin production may be carried out in the presence of SH group blocking substances such as iodine acetate. In this case, the proportion of fibrin in the form of fibrin monomers increases.

In addition to the above, depending on the application, a material with a large amount of crosslinking in the lateral direction may be desired. This is especially the case when the dry formulation requires a particularly high fibrin content of 80% by weight or more. If the proportion of lateral crosslinking is high, the structure becomes tighter and the residence time of the dry formulation becomes longer when it is introduced into the tissue. When attempting to increase the degree of crosslinking in the lateral direction, the total reaction time after addition of thrombin is extended to 40 minutes or more, and in some cases up to several hours.

The present invention also includes a dried preparation obtained by freeze-drying the tomodal obtained from this. In this case, it is desirable to prevent the ripple structure from being destroyed. Immediately after addition of thrombin, the mixture is therefore placed in a lyophilizer, kept stationary at room temperature during the defined reaction period, and immediately frozen after the reaction.

Furthermore, the crosslinking of fibrin polymers can be controlled by the addition of crosslinking agents. Suitable crosslinking agents include, for example, divalent crosslinking agents such as 5PDP (enuscunnimino-3-(2-pyridylditeo)-glupineto), or formaldehyde, glutaraldehyde, malonyldialdehyde, gamma nobic acid and/or derivatives thereof. Divalent organic substances that react with aminoglugs to form peptide bonds, such as eg, divalent organic substances, are discussed.

Since the cross-linking agent has limited performance, its addition chamber is at 0% of the total mixture.
It is possible to lower the power by 1 to 5%.

For example, if all three glutaraldehydes are added to a 100M mixed solution, a result suitable for the percentage can be obtained. Such crosslinking agents also increase the tensile strength and breaking strength of the resulting dry formulation.

In this way, according to the present invention, fibrin is formed on the spot from the water bath solution containing fibrinogen and thrombin and is included in the dry preparation.

f't7'C fibrin produced by this method is separated from human plasma by Caα2 precipitation method to produce fibrin.
Compared to P 3.523.807, it is more natural, has better solubility, and has higher purity. The latter fibrin is practically insoluble and contains other plasma proteins, making it less suitable for use as a wound dressing. .

When producing fibrin gold in situ, it is preferable to set all conditions so that the resulting fibrin polymer has covalent bonds between adjacent fibrin molecules. For this purpose, the reaction mixture must contain at least a catalytically active amount of factor (2). Preferably 111L
1 of the reaction mixture shall contain at least 0.5 to 1 unit of factor. What is particularly appealing about this fibrin polymer is that its covalent bonds primarily result in the formation of longitudinal crosslinks; in other words, the total number of covalent crosslinks is lateral. The share &j allocation related to crosslinking must be less than 2%.

Glue electrophoresis can be used to examine the extent of lateral crosslinking. Particularly preferred fibrin polymers are those in which the fibrin is primarily crosslinked in the longitudinal direction by di-linkage of gamma-linkages. The primary composite can be made by delayed polymerization and/or crosslinking.

When fibrin production, polymerization, and crosslinking have sufficiently progressed, the reaction mixture is deep frozen.

For this purpose, rapid freezing is carried out, bringing the temperature within 5 minutes to -20°C, preferably below -40°C. The choice of cooling temperature cannot be said to be extremely important. This is because thrombin and other coagulation factors do not substantially exhibit any noticeable activity below 0°C.

The material is maintained at the cooling temperature until the entire mixture becomes a completely solid frozen mass. It has been proven that depending on the amount of liquid, it is optimal to retain the liquid in the freezing @mold at 40°C.

A drying process is then carried out under known conditions. For this purpose, the entire frozen body is placed in a vacuum chamber and heated gradually, so that the evaporating liquid is constantly evacuated and deposited in a cooling trap, so that the deep frozen body never liquefies.

If the degree of vacuum is 1 to 60 Pa or less, the drying work will be approximately 3 to 60 Pa.
It should be finished in 8 hours.

The product thus obtained is a fluffy, soft mass exhibiting a foamed, felt-like or wool-like structure.

When observed using a microscope at 200x magnification, it can be seen that the thin fibers are intertwined with each other in a net-like pattern.

The resulting woolly material has a volume equal to that of the initial liquid mixture and has a uniform, homogeneous composition t-i within the entire woolly material. In most cases, the specific gravity of the wool-like structure is between about 0.1 and 0.5 r/clI, and if a fluffier and lighter material is required, the solution can be foamed before filling into freezing molds. Just let it happen. For this purpose, an inert propellant such as nitrogen or carbon dioxide is blown into the liquid.

If the foam pore size is predetermined, surfactants can be added to articulate it.

The external dimensions of the wool obtained after freeze-drying are tailored to the purpose of use.

If the dry preparation is used as a reservoir for the active ingredient or is inserted inside the tissue, spheres with a diameter of 1 to 3 c7n, cubes with a side length of 1 to 3 cIIL, or of similar dimensions. It has been found that it is particularly suitable for use in the form of dandruffs, suppositories and the like. In order to obtain a product with such external dimensions, the entire mixed solution may be filled into a freeze-drying foundry having such a shape.

Once the drying process is complete, the dried formulation is pulled out of the mold by carefully loosening one end of the bottom. Temporarily place the resulting wool-like material on aluminum foil and cut it to desired dimensions as necessary.
Place in a dented glass container and cover with aluminum foil to seal.

Next, the nine containers containing the contents are irradiated with, for example, X-rays (irradiation amount:
Sterilize at 3000 rad for 3 minutes). The product is moisture protected and the sterile package can be stored at room temperature indefinitely without significant loss of activity.

As already explained, there are many opportunities to use the dry formulation according to the invention, and furthermore the composition of the dry formulation can be varied depending on the respective application.

According to the most general embodiment of the invention, the woolen material is
In addition to at least a catalytically active amount of thrombin,
about 10-95 weight percent fibrin, about 5-90
% by weight of fibrinogen.

Here, the term "at least catalytically active cap" refers to about 0.1 to 10 units, preferably 3 to 10 units per cIi of wool-like material.
This means an amount of thrombin of 8 units. In this case, the "unit" is "one unit of NrH" (American National
This is the standard of the 1st Institute of Health) and is commonly used among experts in this field. If the amount of fibrin is less than 10% by weight, even a dry preparation exhibits strong characteristics of fiprinodan, and the product is brittle and has insufficient mechanical strength. Therefore, the amount of fibrin should be at least 10 weight percent, preferably 30 weight percent or more, based on the weight of the dry formulation. When the fibrin volume is greater than 95 weight percent, such materials are less likely to be catabolized by the organism without the need for reaction conditions that produce solids with physiologically low activity. Therefore, the amount of fibrin should not exceed 1 95 ftft %, and should not exceed 70 ft ft % of the weight of the wool. Dry formulations containing about 20 to 70 cents of fibrinogen and about 80 to 70 cents of fibrinogen become particularly similar and compatible with natural wound closure materials when exposed to body fluids and absorb moisture, and are therefore particularly preferred. .

Powdered biochemical substrates suitable for promoting hemostasis and providing optimal biochemical control of wound closure are described in detail in European Patent No. 81110615, 2, dated December 18, 1981. This can be applied in powder form over the preformed dry formulation according to the invention.

If necessary, the contents of this European patent application can be incorporated into the present specification by reference. This powdered biochemical matrix is designed for optimal activation of the intrinsic and/or extrinsic coagulation system and taking into account various physiological and pathological factors. This substrate contains inter alia fibrinogen, components of the thrombin/prothrombin complex, and protease inhibitors. Depending on the application, platelet extract, antibiotics, and others may be added in an appropriate mixing ratio. According to a preferred embodiment of this plasma derivative, its substantial composition is 80-94 weight percent fibrinogen, 1-94 weight percent fibrinogen;
10] ijl percent thrombin and/or prothrombin, 0.01-3 weight percent fibrinolytic inhibitor, 0.4 weight percent or less cryogenically insoluble globulin, and additionally phospholipids, prostaglasin. ,
Dessicants, stabilizers, antibiotics and/or blood clotting factors may be included. The last additional substances are all in solid powder form, and the highly potent powder combination of active ingredients is added in a proportion of 0.1 to 5 parts by weight per 100 parts by weight of the dry formulation. . These small amounts of active ingredient are preferably blown onto the surface of the dry formulation in a stream of sterile gas to ensure sufficient coverage. The fibrin/figurinodan wool-like material thus obtained can be used directly for the treatment of wounds, or can be incorporated into a bandage (with pansoukola) for first aid.

In this way, it is possible to obtain a wound dressing containing a highly potent active ingredient gold complex with hemostatic promotion and optimal wound closure biochemical control action on the surface of a pure biological support material. I can do it.

Furthermore, the dry formulation according to the invention can also be used as a support material. It can also be used as an active ingredient reservoir for one or more active ingredients to enhance the therapeutic effect. For this purpose, the dry formulation must be composed primarily of fibrin. The high fibrinocap ensures that the woolly material has high mechanical strength, sufficient residence time even in tissues and total release of the active substance over a prolonged period. Additionally, ingredients such as collagen can be added to control the release time and amount of the active ingredient. The release of the active ingredient is also influenced by the type and degree of fibrin crosslinking. For this purpose, the support material should be comprised of about 65 to 95 weight percent fibrin and about 5 to 35 weight percent fibrinogen, and additionally contain at least one active ingredient to aid in the therapeutic process. Approximately 70
~85 weight 1% fibrin and about 15-30 ml
Particularly good results have been obtained with a support consisting of % fibrinogen.

The active ingredient can be introduced beforehand into the solution used to produce the dry formulation, and it is also possible to freeze-dry it at the same time. Alternatively, it may be lyophilized and subsequently introduced into a dry preparation.

Thus, the present invention provides a support system comprising a fibrino-mixed foam. It supports biochemical agents and substrates for promoting hemostasis and optimal biochemical control of wound closure. The basic material of this support system is a foam composed of a mixture of fibrin and other materials. Depending on the purpose of use, fibrin, thrombin, prothrombin, platelet extract, protease inhibitor, etc. are added to the fibrin mixed foam individually or in a desired combination at an appropriate mixing ratio. Mixtures of proteins, such as albumin, lipoproteins, fibrin,
Fibronectin, globulin, etc. allow adjustment of the desired flexibility and stability of the foam. Depending on the purpose of use,
In particular, fibrin, thrombin, gluthrombin, platelet extracts, protease inhibitors, antibiotics, etc. may be added to the fibrin mixed foam individually as a mixture or in any desired combination or appropriate mixing ratio. Additionally, the fibrin-mixed foam can be supplemented with a matrix or matrix admixture for promotion of hemostasis and optimized chemical control of wound closure.

EXAMPLES The following examples are intended to explain the present invention in more detail and are not intended to limit the scope of the invention.

Example 1 5? Fibrinogen (as explained above, a microcrystalline preparation obtained from human plasma by alcohol/glycine precipitation) was dissolved in 100% 0.9% Naα solution so that the final protein concentration was 50η/d. do. 300 units of thrombin ([Tostasyn J] from Hofmann-Lalotsch, Glendetshaveen) are added to the fibrinodef solution while stirring. Stirring is carried out briefly and then the entire mixture is poured into freeze-drying molds. Hold at room temperature for approximately 20 minutes. Next, deep freezing treatment is performed. That is, the entire mixed solution together with the mold is cooled to about -401: within 10 minutes. Subsequently, the formed frozen bodies are freeze-dried. For this purpose, the vapor phase is continuously discharged under vacuum conditions and cryo-separated. In this way, a mixture of fluffy, continuous protein wool-like fibrinogen and soluble fibrin is obtained. This mixture has good mechanical strength and breaking strength. This dry formulation contains, in addition to a catalytic amount of thrombin, about 2
It is composed of 0 weight percent fibrino and 80 weight percent fibrinogen.

The following tests were conducted on this dry preparation.

Measurement of fibrin content: 1.0 t of dry preparation i10+11j is added to a 0.9% Naα solution and stirred to remove insoluble components. Add the supernatant liquid9.
Sodium lauryl sulfate (mercagtoethanol is not added) is added to this, and fibrin and fibrin monomer fiprinodan are separated using r-lumi electrophoresis. After staining with oomassie brilliant glue, the component allocation is determined by photometry.

The results showed that the fibrin content was about 20% and the fibrinogen content was about 80%.

Determination of fibrinogen activity 3 units of thrombin are added to 1 ml of the fibrinogen solution obtained by the above method. The generated fibrin is wrapped around a rod and separated from the rest of the liquid. The absorbance before and after fibrin separation is measured using a photometer. Based on the measured values, calculate the percentage of fibrinogen that can be clotted. In this figurinodan, it is more than 85% and a friend.

Measurement of thrombin activity Take 1f of the dry preparation and place it in 10 HLt of 0.9% Naα solution and keep in a greenhouse at 37°C. Measure the time until the onset of coagulation. A reference solution containing pure fibrinogen and increasing amounts of thrombin is prepared and used as a standard for the determination of thrombin activity in the lyophilizate. In this case, about 35~
We were able to detect 45 units of thrombin.

Example 2 Take 9t of fibrinogen t-IQQd in distilled water,
Add 300 units of thrombin to this. After pouring the solution into the freeze-drying mold, observe the progress of the reaction,
If the viscosity increases significantly after about 20 minutes, immediately deep freeze the material. By increasing the amount of thrombin added, the percentage of soluble fibrin increases to 40-50%.

Example 3 Fibrinogen tlo01 of 91! j's 0.1% cola
1 million units of Rantamaicone are added to the solution. Additionally, 100,000 units of trasilol ('
prasylol) Add to the f solution. Like this! I4
! Add 3000 units of thrombin to the mixture prepared in A while stirring, and pour into a freeze-drying mold. After 6 hours of reaction, the contents of the mold are deep frozen and lyophilized.

Example 4 5f fibrinogen tloOj17 dissolved in 1% albumin solution, 3 million units of gentamicin, 500
,000 units of trasylol and 3,000 units of thrombil are added. Twenty minutes after pouring into the freeze-drying mold, 2-dartaralde zeta is added as a crosslinking agent. and lyophilize the mixture.

Example 5 5 t of fibrinogen is added to 100 - of plasma and dissolved under stirring. 300 units of thrombin are added, and after 20 minutes, the material that has become rolyzed is placed in a freeze-drying mold, deep-frozen, and then freeze-dried.

Example 6 51 Fiprinodan? 100 units of thrombin is dissolved in 0.9% Naα bath solution, and 3,000 units of thrombin is added thereto. After 12 hours, the fibrin is stirred to convert from a glue shape to a fiber shape.

The material in the form of fibers is suspended in a 5% albumin solution and its dimension f: mechanically reduced.

After adding 2d of glutaraldehyde, the admixture is deep frozen and then lyophilized.

Claims (12)

[Claims] (1) In dry fibrinogen-containing preparations that have foam-like and wool-like structures obtained by freeze-drying and are used in particular as a support for active ingredients, in addition to at least a catalytically active amount of thrombin,
A fibrinogen-containing dry formulation, wherein the wool-like material consists essentially of about 10 to 95 weight percent fibrin and about 5 to 90 weight percent fibrinogen. (2) 1 cm^2 of wool-like material is approximately 0.1 to 10 units (
Claim No. 1 containing thrombin (NIH unit)
) Dry preparations described in section 2. (3) A wool-like material primarily for use as a support material,
about 65-95 weight percent fibrin and about 5-35 weight percent fibrin
A dry formulation as claimed in claim 1 or 2, further comprising at least one weight percent fibrinogen and additionally at least one healing-promoting active ingredient, such as an antibiotic. (4) The dry preparation according to claim (3), wherein the wool-like material additionally contains a component that controls the time and amount of release of the active ingredient. (5) Fibrin is essentially fibrin monomer γ
- Claim No. 1 (1) connected only through chain
) to (4). (6) Claims (1) to (5) wherein the wool-like material additionally contains one or more glycoproteins, namely albumin, lipoprotein, fibronectin, or globulin.
Dry preparation according to any of paragraphs. (7) The dry preparation according to any one of claims (1) to (6), wherein the wool-like material additionally contains one or more fibrinolysis-inhibiting substances. (8) A method for producing a dry preparation, characterized in that fibrin is produced in situ in an aqueous solution containing fibrinogen and thrombin, and the resulting reaction mixture is deep frozen and freeze-dried. (9) a) adding fibrinogen to water, human serum or a saline solution; b) adding, if desired, one or more active ingredients, additives, etc. to the fibrinogen-containing solution; and c) obtaining thrombin or a precursor for thrombin generation. d) keep the mixture under normal conditions to allow the reaction to proceed until some, but not all, of the fibrinogen reacts to form fibrin monomer; e) deep freeze once the reaction has proceeded sufficiently. and f) finally freeze-drying. (10) 10 to 90 m/ml for the initial solution
g of fibrinogen and 0.1 to 10 units of thrombin are added, stirred at room temperature for 10 to 40 minutes after the addition of thrombin, and the reaction mixture is cooled to a temperature below -20°C, and the resulting frozen product is transferred to the liquid phase. The method according to claim 8 or 9, wherein the method is freeze-dried without forming a new one. (11) Any of claims (8) to (10), wherein the production of fibrin is carried out in the presence of a substance that sequesters SH groups and/or the mixture of fibrin is crosslinked by the addition of a crosslinking agent. Method described in Crab. (12) Any one of claims (8) to (11), in which the dry preparation taken out from the freezing mold is cut into molded shapes, moisture-proof packaged, and the package is sterilized together with its contents. The method described in.