Biological adhesive glue
(57) Abstract:The invention relates to the field of medicine and relates to adhesive compositions to stop bleeding on the basis of factor VIII. The essence of the invention lies in the fact that the proposed composition contains cryoprecipitate obtained by refrigerated centrifugation at a speed of 2500-3000 rpm for 1-1 .5 h, the dose of which contains factor VIII activity from 150 to 220 units , thrombin activity from 160 to 700 units in 7-15 ml of isotonic solution of sodium chloride and 3-10 ml of 10% calcium chloride or calcium gluconate. The technical result is the creation of adhesive compositions, the application of which allows you to stop bleeding not only from the capillaries, but vessels of small and medium caliber, and extensive tissue surfaces. The invention relates to the field of medicine and relates to adhesive compositions based on fractional plasma protein (factor VIII) to stop the bleeding and restore the biological integrity of tissues (reconstructive and trauma surgery, traumatology, emergency medicine, obstetrics, gynecology, ophthalmology, otolaryngology (ENT).Adhesive compositions based on biological substances, ear surgical field;
- slow tissue regeneration;
- toxic effect on the surrounding tissue.Fibrin glue is not only stops the bleeding but also promotes tissue regeneration due to the unique stimulating properties of fibrinogen, a multifunctional protein, which serves as a matrix for the regulation of fibrinolysis, attachment of the cells in the repair process, activates the production of leukocyte germ angiogenic factors.Known fibrin-containing adhesives, manufactured by companies like drugs Beriplast (Germany), Tissucol (Austria) , Biocol (France) .They are similar in composition. For example, glue Beriplast consists of: 1) lyophilized fibrinogen human blood 115-232 mg; 2) thrombin (bovine) 400-500 units; 3) placental protein fraction containing 40-80 units of factor XIII; 4) solution of Aprotinin - up to 1000 KE; 5) solution of calcium chloride, 40 mmol/l 25 ml.The development of such domestic product is complex due to the lack of technology for factor XIII, no drug Aprotinin and the high cost of the final product.The closest in technical essence and the achieved result is fibrin glue, developed in the PCR CCH 7 Moscow . His Doc is t in the usual way, to it was added a solution of PEG - 6000 and 1 ml of aminocaproic acid (sterile solutions seamed in sterile pharmacy ware, pull through a puncture of the tube by vacuum aspiration).2). The vial is shaken, defend for 30 minutes and centrifuged for 15 minutes at 4oC.3). The supernatant is sucked off, and the residue is dissolved in sodium citrate and collect in a sterile rolled penicillin vial (about 4 ml of the fibrinogen content of the last 40-60 g/l) and additionally paraffinicity. For polymerization on the bleeding surface to it add 500 units of thrombin in 4 ml of 10% solution l2.The disadvantages of the adhesive obtained in this way are:
- the possibility of contamination of the plasma by viruses of different origin, including AIDS and hepatitis C;
- the complexity of the composition of the first component adhesive consisting of cryoplasty, PEG - 6000 and - aminocaproic acid;
- the use of expensive sodium citrate for pH regulation;
- narrow the scope of its application: to stop bleeding only from the capillaries;
- receiving adhesive directly on the wound surface, alternately introducing the components of the adhesive composition is LASS="ptx2">The present invention is the creation of a fibrin-containing safe glue based keratinizing cryoprecipitate, which eliminates viral contamination. Glue produced from domestic raw materials and it is designed to stop bleeding from parenchymatous, hollow organs, extensive bleeding tissue surfaces and vessels of small and medium caliber, to commit their own biological tissues (autotransplants) and to improve reparative processes in the application of the adhesive mass.The solution of the problem is the creation of a biological adhesive glue (LHC), including cryoprecipitate, in the amount of 1 dose containing factor VIII activity from 150 to 220 units, thrombin activity from 160 to 700 units in isotonic sodium chloride (7-15 ml) and calcium chloride or calcium gluconate (3-10 ml).Preferably the TANK contains: thrombin activity from 240 to 560 unitsPreferably the TANK contains factor VIII activity from 160 to 200 unitsThe advantages of the present invention:
1. Use keratinizing cryoprecipitate, which eliminates viral contamination.2. Ease of access (the user.4. Economic benefit.5. Use to stop bleeding not only from the capillaries, but also vessels of small and medium caliber, and extensive tissue bleeding surfaces.6. Reliable acceleration and stimulation of tissue regeneration.7. The possibility of applying for the manufacture of biological prostheses, since the adhesive composition "in vitro" is able to continuously retain a given shape and fully biologically compatible with body tissues.8. The ability to maintain in vivo in humans adhesive properties within 5-7 days, forming on the damaged surface of the fibrin film, followed by granulation and epithelialization.The invention is illustrated in the following examples.EXAMPLE 1.To obtain cryoprecipitate karantinirovaniya (within 6 months) fresh frozen plasma (donor or AUTOLOGOUS) is thawed at room temperature to form a "wet sugar", in the amount of 300 ml, then plasma substance centrifuged in a refrigerated centrifuge for 1-1 .5 hours at a speed of 2500-3000 rpm the Supernatant part of the plasma (supernatant) and the average fraction of plasma (concentrate NAT is ATiM sediment (cryoprecipitate) leave it in the main bag and freeze again. Received cryoprecipitate contains factor VIII activity from 150 to 220 units Store cryoprecipitate at a temperature of from 28 to 73oC.EXAMPLE 2.To obtain biological adhesive glue:
1. One dose of cryoprecipitate containing 160 units of antihemophilic globulin (factor VIII), thawed at room temperature under the conditions of shaking until a homogeneous mass (to avoid the formation of lumps).2. Thrombin, taken from activity in the amount of 240 units, dissolved in equal amounts in the two test tubes containing 10 ml of isotonic NaCl solution and 5 ml of 10% l2respectively, then mixed.3. Cryoprecipitate and thrombin solution are mixed immediately prior to use.EXAMPLE 3. To obtain biological adhesive glue:
1. One dose of cryoprecipitate containing 160 units of antihemophilic globulin (factor VIII), thawed at room temperature under the conditions of shaking until a homogeneous mass (to avoid the formation of lumps).2. Thrombin, taken from activity a quantity of 700 units, dissolved in equal amounts in the two test tubes containing 10 ml of isotonic NaCl solution and 5 ml of 10% gluco is directly before use.EXAMPLE 4.Differs from example 2 in that cryoprecipitate is taken from activity antihemophilic globulin (factor VIII) 180 unitsEXAMPLE 5.Patient R. , 75 years enrolled in the MSC 1 ZIL with clinical signs of gastrointestinal bleeding and low hemodynamic indices in three days from the onset of the disease. When emergency gastroduodenoscopy revealed extensive (up to 3.5-4 cm) ulcer of the gastric body with blood clot under which actively received fresh blood. The situation is regarded as an ongoing gastrointestinal bleeding (Forrest 16). Due to the fact that the ulcer was located on the lesser curvature of the stomach, it has not been technically possible to perform coagulation and injection methods of stopping bleeding, and the risk of emergency surgery was very high in patients of advanced age with severe concomitant somatic pathology. It was decided to treat the surface of ulcers Tank. Two component adhesive were mixed ex tempora and applicaany through the catheter on the ulcer surface. Applied composition, filling ulcerative defect, formed matte gel fibrin film, intimately adjacent to the mucosa. Already during manimala, where she conducted infusion-transfusion correction of blood loss circulatory disorders in combination with antiulcer drug treatment. When the control gastroduodenoscopy 24 hours bleeding is not identified, ulcerative defect was still filled with fibrin film. After stabilization of the patient was transferred for further treatment in the surgery Department, where she continued combination therapy. After three weeks at endoscopy ulcer defect is represented by a red scar. In a satisfactory condition the patient was discharged home.EXAMPLE 6.Patient K., aged 65, was admitted to the vascular Department of MSU 1 ZIL with a diagnosis of atherosclerosis of the lower extremities, Leriche syndrome, chronic arterial insufficiency stage 2B. During angiographic examination, it was found 50% stenotic disease aortic aneurysm and occlusion of the ileo-femoral segment on both sides.In order to restore blood flow in the lower extremities, the patient was shown reconstructive surgery of the aortic-bifemoral bypass. In the process of operation was used vysokoporodny Dacron prosthesis "North". To minimize blood loss n in the TANK before implantation for 5 minutes. According to our data, the estimated blood loss without the use of the TANK enabling blood flow ranged from 600 to 900 ml, which subsequently caused various complications. When using TANK hemorrhage along the line of anastomosis and through the pores of the prosthesis was not observed. The patient was discharged 10 days after surgery in satisfactory condition.Sources of information
1. Gastpar h Local application of human fibrin seal (Tissucol/Tissel) in tonsillectomies and adenectorrues in patients with bleeding disorders. Ten years clinical experience // Fibrin sealant in operative medicine / Shlag G. Readi H (Eds). - Springer Verlag: Berlin - Heidelberg, 1986. - P. 128-131.2. Kraum H. B., Nathan R. C, MacKable J. R. Clinical use of noneautologous fibrin glue // Amer. Surg. - 1988. - Vol. 54, 9. - P. 570-573.3. So Century. Horobryh, A. N. Antonov, G. K. Orlov, I. M. Soloviev. Receiving fibrin glue in clinical conditions //M - 1994. Scientific achievements into practice, vol. 7, pp. 133-136. Adhesive composition to stop the bleeding and restore the biological integrity of tissues, characterized in that it contains cryoprecipitate obtained by refrigerated centrifugation at a speed of 2500-3000 rpm for 1-1 .5 h, the dose of which contains factor VIII activity from 150 to 220 units, thrombin activity from 160 to 700 units in 7-15 ml of isotonic solution of sodium chloride and 3-10 ml of 10%
FIELD: medicine, hematology, pharmacy.
SUBSTANCE: invention relates to the composition of factor VIII composed without addition of albumin and comprising the following excipients of composition in addition to factor VIII: from 4% to 10% of filling agent taken among group consisting of mannitol, glycine and alanine; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance about between 6 and 8. Alternatively, the composition can comprise from 2% to 6% of hydroxyethylstarch; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance between 6 and 8. In additional variant of realization of invention the composition can comprise: from 300 mM to 500 mM of NaCl, from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose and arginine; from 1 mM to 5 mM of calcium salt, and buffer agent. The composition provides stability in the absence of albumin or other proteins.
EFFECT: valuable properties of compositions.
35 cl, 11 tbl, 7 ex
FIELD: pharmaceutical industry and biotechnology.
SUBSTANCE: method consists in the following: kryoprecipitate of donor blood plasma is suspended in heparin solution, resulting suspension is consecutively treated with aluminum hydroxide and polyethylene glycol-400 to remove ballast proteins until content of aluminum hydroxide in solution achieves 0.3% and that of polyethylene glycol-400 2%. Thus treated material is subjected to viral inactivation by solvent-detergent technique in presence of Tween and microfiltration followed by chromatographic fractionation on DEAE-containing carrier using buffer solutions, pH 6.75-6.85, with different ionic forces. Resulting concentrate of factor VIII is stabilized with albumin solution to provide final albumin concentration in the preparation 0.1%, sterilized by microfiltration, and transferred into lyophilized form, in which viruses are additionally inactivated via heat treatment.
EFFECT: preserved specific activity of factor VIII with high degree of purification and increased storage stability.
5 cl, 1 tbl
SUBSTANCE: invention relates to methods for preparing biologically active substances from donor blood. Method for enriching donor blood plasma cryoprecipitate with factor VIII involves defrosting freshly frozen donor blood plasma at +1-4°C to obtain cryofractions from plasma cryosupernatant and cryoprecipitate. After formation of the donor blood freshly frozen plasma dry calcium chloride is added in the amount from 0.001 to 0.004 mole/l followed by addition of sodium citrate in the amount from 0.05 to 0.10 mole/l and stirring at +1-4°C for 10-15 min. Then prepared cryoprecipitate deposit is separated by centrifugation. For preparing cryoprecipitate freshly frozen plasma is used prepared by usual method with using conventional preserving agents. Using the method provides significant increasing the content of factor VIII in cryoprecipitate being without appreciable co-precipitation of inert proteins.
EFFECT: improved enriching method.
2 tbl, 3 ex
FIELD: medicine, pharmaceuticals.
SUBSTANCE: invention relates to pharmaceutical agent containing factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide. Disclosed are application of factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide in production of drug, kit for episodic bleeding treatment, as well as methods for prophylaxis and treatment of episodic bleeding in subject.
EFFECT: accelerated coagulation of FVIII-deficit plasma.
43 cl, 1 tbl, 2 dwg, 6 ex
FIELD: medicine, pharmacy.
SUBSTANCE: invention relates to a novel stable ready formulation of pharmaceutical composition containing VIII factor and can be used in treatment of hemophilia. Invention relates to a solid pharmaceutical composition prepared by lyophilization of an amino acid-free solution and comprising the following components: (a) VIII factor in the concentration 50-10000 IU/ml for human VIII factor or human recombinant VIII factor, or 50-10000 U/ml for swine VIII factor or swine recombinant VIII factor; (b) surfactant in the concentration above critical micellar concentration up to 1% (vol./vol.); (c) calcium chloride; (d) sucrose; (e) sodium chloride; (f) trisodium citrate, and (g) amino acids-free buffer in the concentration 1-50 mM with pH 6-8 before lyophilization and after dissolving in water for injection. Also, invention relates to a liquid pharmaceutical composition prepared after dissolving indicated stable solid pharmaceutical composition in sterile water optionally containing sodium chloride. Invention provides preparing a stable ready formulation of pharmaceutical composition of VIII factor wherein albumin is replaced with other stabilizing agents.
EFFECT: improved and valuable properties of pharmaceutical composition.
25 cl, 3 tbl, 3 ex
FIELD: technological processes.
SUBSTANCE: preparation of human blood coagulation factor VIII is made by producing cryoprecipitate out of blood plasma, virus inactivating treatment, purification of cryoprecipitate solution, concentration, sterilizing filtration, bottling and drying. At the same time cryoprecipitate is produced with the help of running water with the temperature of (4±2)°C during unfreezing of fresh frozen plasma, virus inactivating treatment is carried out by means of solvent-detergent method, chromatographic purification of virus inactivated cryoprecipitate solution is carried out with application of sorbent with fractionation interval up to 20,000 kilodaltons, at the flow rate of (20±15) cm/hr, with further concentration by means of ultrafiltration on hollow fibers. Stabilizers are added, such as albumin in final concentration of (5±3) g/l, sugars and amino acids up to final concentration of (10±3) g/l. During drying initial preparation temperature is (25±15)°C below zero, final temperature is (25±8)°C, total duration of preparation drying process is (45±7) hours.
EFFECT: coagulation factor yield stability is increased and technological production design is simplified.