TW449481B - Method for producing fibrinogen particles suitable for intravenous injection - Google Patents

Abstract

The present invention relates to a method for producing a protein particle that can reduce loss of blood in a surgery or medicine treatment and can accelerate a medicine treatment and the recovery speed of an operation, particularly fibrinogen particles suitable for intravenous injection. The protein particles suitable for intravenous injection according to the present invention is produced from human albumin according to the following process: preparing a protein particles suspension, in which proteins powder is dissolved in buffer under suitable osmotic pressure; adding surfactant into the protein solution; adding cross-linking agent into the solution of the protein and the surfactant; adding an alcohol solution into the mixture of the protein, the surfactant and the cross-linking agent, thereby producing a fibrinogen particle suitable for intravenous injection.

Description

449481 V. Description of the invention (1) The present invention is related to-it can reduce surgery and medical treatment

Protein I that bleeds and can accelerate medical treatment and surgical recovery, especially the manufacture of fibrinogen particles suitable for intravenous injection. It is well known that human blood is the foundation of health. Many surgical and medical treatments, such as cancer surgery, will Causes blood loss, but blood transfusion is a very complicated process that may cause AIDS and the introduction of hepatitis virus: In addition, especially for patients with platelet deficiency, excessive blood loss is even greater. Therefore, a substance that is helpful for coagulation is urgently needed in the clinic. These problems are mentioned above. Since these substances are as effective as platelets, they can reduce blood loss in patients undergoing surgery and surgery and patients with platelet deficiency ’, so after injection, they must have certain properties. One: the diameter of the human microvessels is 7 microns, then the diameter of the substance particles must be less than 7 microns; the second: the substance particles themselves should not agglomerate in the blood stream, but they agglomerate at the wound, the substance particles will not cause blood cells to lie in the blood vessels It will take part in the coagulation, but it will participate in or promote blood cell aggregation at the wound. Particles of such substances help blood to aggregate, so the particles must have surface molecules, such as fibrinogen, that allow them to participate in blood coagulation. In current technology, such as K. j. Widder and others in "Pharmacology and Chemotherapy Run Development", Vo 丨. 16,213_271, anti-cancer agent biophysical display water, #magnetic tongue steep microsphere Zhao and other carriers 〃 As mentioned in the article, protein microspheres are synthesized by using protein body Γ = and an emulsion obtained by heating or polymerizing the oil ^, and because the protein is easily deteriorated in this process, the microparticles have a short agricultural period. Therefore, in actual treatment, it needs to be able to be stored in blood vessels to

4 4 9 48 1 V. Description of the invention (2) Particles that do not deteriorate in one day. 0? 611116111 et al. In U.S. Patent No. 1,84,107,288 (U.S. Patent No. 1) 84, 107, 288 (Effective extremely fine particles in injection compositions and production methods thereof) proposed another method: wherein the concentration of the desolvating agent is adjusted by using an opposite solubilizing agent and The protein solution was brought to a desolvation limit. The problem with this method is that the conditions at the beginning of the reaction are not stable 'and the polymerization is accompanied by a large amount of polymerization which leads to the formation of spheres. The diameter of the condensed sphere is larger than 7 micrometers, which exceeds the diameter of the blood vessel ', which can cause vascular occlusion and cause danger. However, the actual treatment of cloth requires particles that do not agglomerate much smaller than 7 microns in diameter. An object of the present invention is to provide a method for producing fibrinogen particles suitable for intravenous injection. In the present invention, the spheres formed in the presence of a crosslinking agent have an average diameter of less than 1 micron and a small coefficient of variation. In the present invention, 'fibrinogen can be used as a longitudinal molecule, which can carry protein spheres to the wound to reduce the amount of bleeding. In the present invention, 'as the effect of this composition is coagulation, and its efficacy is the same as that of platelets, it is also called 〃coagulation plate〃 or 〃platelet substitute or hemostatic plate " (f ibrinop 1 ate' p 1 ate 1 et aggregat i on substitute). In the present invention, the composition of this protein is (in parts by weight, human albumin: 90-100 parts, fibrinogen: 0.02-6 parts, and the composition of the present invention is a protein synthesis made by the following improvements

V. Description of the invention (3) * »1 · First make a protein particle suspension 'where: a.' Dissolve the protein powder in a warm flush with an appropriate osmotic pressure; b. Add the surface activity to the protein solution described in a ' C. Adding a cross-linking agent to the protein and surfactant mixture described in b; d. Adding an alcohol solution to the protein, surfactant, and cross-linking agent described in c. Immediately, a turbid suspension of protein spheres appeared, which was suitable for intravenous injection and did not block blood vessels. In the present invention, the protein solution is a human albumin solution. In the present invention, the surfactant is a detergent. In the present invention, the surfactant may be selected from one of Sodium tetradecyl sulfate 'Tween-80' TritonX-151: In the present invention, the cross-linking agent molecule includes glutaric acid Covalent small molecules of glutaraldehyde * In the present invention, the buffering pH ranges from 4 to 8. In the present invention, the osmotic pressure range is 14-68OmOsm «2. After the above-mentioned suspension turbidity appears, the surface protein is added to the sphere in the suspension, so that the unreacted part of the sphere will be passed through a covalent bond. The surface protein may be fibrinogen. 3 'The above-mentioned sphere suspension of human albumin to which the surface protein has been added is further processed into a powder. The process includes dilution, filtration, dialysis,

^^ 9481 V. Description of the invention (4) The data were reconstituted by vacuum drying, freezing, and powder separation after centrifugation, electrophoresis, and column separation. In this process, a buffer solution needs to be added, or a biological solution such as a buffer solution and a preservative must be added. These trial types and dosages are well known to those skilled in the art. Therefore, a method for manufacturing fibrinogen particles suitable for intravenous injection in the present invention, wherein the protein particle suspension composition does not cause vascular occlusion (except at the wound site), and the protein particles are passed through human albumin Fibrinogen is formed with an average diameter of less than 7 microns, and is suitable for intravenous injection without causing blood to pass through the human body's white micrometers. In addition, the particle suspension is covalently formed at the wound site, and the body is white again. It is suitable for intravenous injection without causing hemoglobin and fiber. Its surface is fiber. The invention is also a biologically frozen frozen protein particle combination. Material, tube blockage (except at the wound site), the protein cross-links fibrinogen formation, which is flat in addition to the composition of the invention), particle epiprotein, the cold tube blocked vitamin protein of the invention is related to vitamin protein, where the Combination surface, and its average related frozen eggs (except in the original co-prime, is also a kind of the composition is the human body and the particle diameter is smaller than a white particle wound site valence formation part is suitable for the human body veins will not cause white The protein and the surface are 7 micrometers. After biophase composition, the surface of the particles is albumin. After the solution has been reconstituted, the protein particles of the composition are less than 7 in diameter. The injected protein is blocked by blood vessels. Proteinogen, after the internal solution is reconstituted, wherein the composition composition is a human body, and the granules Which were less than straight through

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7 microns. Shoot correction: Another: a new use of the protein particle after intravenous injection is provided, that is, the particle can migrate to a new wound, help blood coagulation and reduce blood loss in patients. In addition, this particle can be used on patients who have trauma, surgery or large, internal and external bleeding, which is extremely beneficial to reduce the blood loss and save the patient's life. The present invention is further described below through examples. Although the following examples use human albumin, other human proteins such as immunoproteins, myoglobin, collagen, and gelatin can also be used and can form spheres. Example 1 Method for forming microparticles without fibrinogen: Put human albumin powder into a 0.9% sodium gas solution, and add a surfactant at a concentration of 0.05 mg / ml to 15 mg / ml, such as tetradecane Sodium sulfate, Tween-80 or TritonX-151 to form a protein solution with a concentration of 5-20% (weight to body weight ratio). Then, 2πΠ diluted with a gasified sodium solution was added to a series of test tubes containing 2 ml of the above-mentioned protein solution containing different concentrations of surfactants. After 10 minutes, 10 ml of ethanol [80% (volume ratio) released with water] was added again, and the solution immediately became cloudy. After another 10 minutes, the suspension was diluted with 5 times the volume of sodium gas solution to observe whether the suspension was clear. The result was negative. Thereafter, the suspension was observed under a microscope at a magnification of 1000, and it was found that the diameter of the particles in the suspension was less than 1 m.

Page 8, 49 48 ____ V. Description of the invention (6) At the same time, it was found that a mixture made of Tri tonX-151 at a concentration of 1-2.5 mg / ml and albumin at a concentration of 100-200 mg / inl best effect. In addition, it was also found that mixtures made with sodium tetradecyl sulfate at a concentration of bu had the smallest coefficient of variation in particle diameter. Example 2 Method for attaching fibrinogen to protein particles as a tracer particle: The same conditions as in Example 1 were used, and the labeled human albumin of Bu 125 was used. The spheroids were precipitated by centrifugation, and the number of radioactive protein particles each contained in the precipitate and the supernatant was measured. It was found that 95% of the albumin particles formed the spheroids. 9% 的 化 化 If the solute of the solution is increased from 0.9% of the concentration of sodium vaporized solution to 2 times', it becomes 1.8% sodium chloride solution or the 109% sodium vaporized solution is reduced to 0.9% of the gasification Sodium solution was found to have little effect on spheroid production. If the pH of the buffer solution is lowered below 4, it is found that the amount of alcohol required to form the sphere is greater than that required when the pH is 8, and the yield of the sphere is slightly increased when the pH is below 4 decline. Example 3 Repeat Example 2 under the most preferred conditions. The difference is that the human albumin is coated with fluorescent thiocyanate. The best conditions of Example 1 are used. In different time intervals after turbidity occurs, Add fibrinogen to the ball suspension

449481 V. Explanation of the invention (7) In the solution, it was found that adding fibrinogen within fifteen minutes after the spheroid formation can stabilize the spheroid and obtain the best mixture. In addition, the spheroid suspension must be further processed 'including dilution, filtration, dialysis, refiltration, centrifugation', electrophoretic separation and column separation, drying by vacuum cooling and cooling and powder reconstitution. During this process_ buffers or preservatives need to be added or changed. According to McGill et al.'S article on j. Lab. Clin. Med. 1987, 109: 127-13, the platelet mold cavity can reduce the bleeding time of platelet-deficient rabbits. The bleeding time of ten rabbits treated with 0mg / kg Busul fan method was 697 seconds. After the injection of 11111/1 ^ fibrinogen-coated spheres (the frozen powder was dissolved with 0.9% sodium gasified solution). ), The bleeding time is reduced to 115 seconds (standard deviation is 43 private). The rabbit ear wound was sectioned and moved under a fluorescent microscope to observe that 'there were fluorescent particles in the blood coagulation. # 果 pairs of platelet deficient immunity = ί fibrinogen spheroid suspension (lmg / ml), bleeding was found 〇 " shortened, no fluorescent particles were found in the ear coagulation, this said: proteinogen is used as a guide molecule Lead particles to the active area of coagulation, and lead to shortened bleeding time and reduce the number of η; trauma or may not have platelet deficiency surgery and have curative effects, feel faster

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Claims (1)

一 "9 扣 j___ VI. Patent Application ^ 1 · A method for the manufacture of fibrinogen particles suitable for intravenous injection, in which the particles are prepared by improved protein in the following ways: a. A buffer suitable for osmotic pressure In the solution, dissolve the protein powder; b. In the protein solution described in a, add a surfactant, the surfactant is one selected from the group consisting of sodium tetradecyl sulfate, TritonX-151 or Tween80; c. In b A cross-linking agent is added to the protein and surfactant mixed solution, and the cross-linking agent molecules are covalent small molecules including glutaraldehyde; d. In the protein, surfactant, and cross-linking agent described in c An alcohol solution is added. 2. The method for producing fibrinogen particles suitable for intravenous injection according to item 1 of the scope of the patent application, wherein the protein solution is a human albumin solution. 3. The item 1 of the scope of patent application The method for manufacturing fibrinogen particles suitable for intravenous injection, wherein the surfactant is a detergent. 4 · The method suitable for intravenous injection according to item 1 of the scope of patent application The method for manufacturing fibrinogen particles, wherein the pH of the buffer solution is 4 to 8. 5. The method for manufacturing fibrinogen particles suitable for intravenous injection according to item 1 of the scope of patent application, wherein The permeation range is 14-680mOsm. 6. The method for manufacturing fibrinogen particles suitable for intravenous injection according to item 1 of the patent application range, wherein the alcohol solution includes ethanol Page 12 449481 Sixth, the scope of patent application --- solution. 7. The method for producing fibrinogen particles suitable for intravenous / main injection as described in any one of items i to 6 of the scope of application for patents, wherein the preparation process further comprises making the protein particle suspension into a powder. 8 The method for producing fibrinogen particles suitable for intravenous injection according to item 7 of the scope of the patent application, wherein the method for improving the protein includes the following steps: separation, drying, and reconstruction. 9. The method for producing fibrinogen particles suitable for intravenous injection according to any one of claims 1 to 6 of the patent application scope, which comprises: adding surface proteins to the sphere suspension. 1 0. The method for producing intravenous fibrinogen particles suitable for the intravenous injection according to item 9 of the scope of the patent application, wherein the surface protein includes human jk fibrinogen. 1 1 * The method for producing fibrinogen particles suitable for intravenous injection according to item 9 of the scope of the patent application, wherein the protein solution is a human albumin solution. 1 2 * The method for producing fibrinogen particles suitable for intravenous injection according to item 9 of the scope of the patent application, wherein the surfactant is a detergent. 1 3 · The method for manufacturing fibrinogen particles suitable for intravenous injection according to item 12 of the scope of the patent application, wherein the method is selected from the group consisting of sodium tetradecyl sulfate, Triton on X-51 or Tween 80 . 1 4 · The method for producing fibrillin protein particles suitable for intravenous injection according to item 9 of the scope of the patent application, wherein the crosslinker molecule is a Page 13 449481 VI. Scope of patent application A covalent reactive molecule including eriodine. 15. The method for producing fibrinogen particles suitable for intravenous injection according to item 9 of the scope of the patent application, wherein the alcohol solution includes an ethanol solution. 116. The method for producing fibrinogen particles suitable for intravenous injection according to item 9 of the scope of the patent application, wherein the pH range of the buffer solution is 4 to 8. '17. The method of manufacturing fibrinogen particles suitable for intravenous injection according to item 9 of the scope of the patent application, wherein the osmotic pressure range is 1 4-6 8 0 m 0 s m. 18. The manufacturing method of fibrinogen particles suitable for intravenous injection according to item 9 of the scope of patent application, further comprising reprocessing the sphere suspension into a powder. 19. The method of manufacturing fibrinogen particles suitable for intravenous injection according to item 9 of the scope of patent application, wherein the protein improvement method includes the following steps: separation, drying, and reconstruction. Page 14