An aqueous solution of factor viii with reduced oxygen concentration
(57) Abstract:The invention relates to medicine, namely to the pharmacy, and relates to a product comprising coagulation factor VIII in aqueous solution with reduced oxygen concentration. The essence of the invention is that the aqueous solution of the coagulation factor may be stored for a long time, if the medicinal product further comprises an inert gas and/or an antioxidant. The technical result consists in increasing the stability of an aqueous solution of coagulation factor VIII. 2 c. and 18 C.p. f-crystals, 4 PL. The present invention tositsa to the final drug product, including coagulation factor VIII in aqueous solution with reduced oxygen concentration. In this form, the activity of factor VIII can be maintained during the period of storage at a very high level. The activity of factor VIII can be maintained over a long period of time if the final remedy includes an optional inert gas and/or antioxidant. The present invention relates also to a method of reducing the concentration of oxygen in aqueous solution and to a method of improving the stability of factor VIII in aqueous solution p. who keeps at least 50% of the initial activity of factor VIII after storage for at least 6 months at a temperature of from 2 to 10% and the pH from 6.5 to 8.5.The background to the invention
The stability of proteins is one of the main problems of the pharmaceutical industry. Often it can be solved by drying the protein through various drying methods such as freeze drying. After that, protein Packed and stored in dry form. The solution prior to drying or drying by freezing, dry material and re-dissolved product must be stable in order to avoid significant loss of activity in the drying process, as well as during storage or application.The freeze drying is an expensive and lengthy process, therefore when making commercial product, it would be highly desirable to avoid this stage. In addition, the patient to use to dissolve the dry protein, which can be uncomfortable.Hemophilia is a hereditary disease known for centuries, but only the last three decades have made it possible to differentiate its various forms: hemophilia A, hemophilia b and hemophilia C. Hemophilia a is the most common form. It affects only men with a frequency of 1-2 persons per 10 000 live new is the first coagulation factor VIII (antiempirical factor), which is a protein normally present in the blood plasma. Clinical manifestation of hemophilia a is a strong tendency to bleeding, and before the introduction of treatment with concentrates of factor VIII life expectancy of these patients were less than 20 years. Concentrates of factor VIII obtained from blood plasma became available during the last three decades. This has significantly improved the situation with the treatment of hemophilia and gave them the opportunity to lead a normal life.Therapeutic concentrates of factor VIII to the present time was obtained by fractionation of blood plasma. Currently, however, there are ways of obtaining factor VIII in cell cultures using the techniques of recombinant DNA, as described, for example, J. Gitschier, etc., Nature 312, pp. 330-37, 1984 and EP-A-160 457. Concentrates of factor VIII derived from blood plasma, contain several fragmented forms with full activity of factor VIII (Andersson and others, Proc.Natl.Acad. Sci. USA, vol. 83, pp. 2979-83, may 1986). The smallest active form has a molecular mass of 170 kDa and consists of two goals 90 kDa and 80 kDa, interconnected ion bridge metal. Reference is made to EP-A-197901. Kabi Pharmacia developed recombinant factor VIII, to the recombinant factor VIII molecule is denoted by r-VIII SQ and is produced by cells of the Chinese hamster ovary (CHO) in culture in the absence in the final passage of serum in the culture medium.The specific activity of r-VIII SQ is about 15 000 ME VIII:C per mg protein.Recombinant factor VIII SQ is indicated for the treatment of classical hemophilia. Dose similar to the dose of plasma concentrates of factor VIII.The structure and biochemical properties of the recombinant products of factor VIII outlines Kaufman in Tibtech, vol 9, 1991 and Hematology, 63, pp. 155-65, 1991. The structure and biochemical properties of r-VIII SQ is described in WO-A-91/09122.Factor VIII fraktsionirovannyj from blood plasma, usually sold in powder form, obtained by drying, freezing, which must be diluted with water.The product containing a small amount of protein, usually loses its activity during purification, sterile preparation, packaging and application. This problem is usually solved by adding albumin human, which significantly reduces the loss of active protein. Albumin human acts as the main stabilizer during purification, sterile preparation and drying by freezing (see review by Wang and others, J. of Parenteral Sci. and Tech. Tom. 42, number 2, Annex 1988). The use of albumin to stabilize factor VIII is known, and currently it is used in all preparations faia of human albumin to pharmaceutical protein, obtained through the techniques of recombinant DNA. In addition, the use of albumin, human as a pharmaceutical excipient often limits the use of many efficient and sensitive analytical methods of studying protein.For the stabilization of the different proteins was proposed several solutions. ER 35204 (Cutter) discloses a method of imparting thermal stability of the protein composition in the presence of a polyhydric alcohol. Further, WO-A-89/09614 (Genentech) discloses a stabilized preparation of human growth hormone containing glycine, mannitol and the buffer, and in the preferred embodiment of the invention also added non-ionic surfactant, such as Polysorbate 80. The nonionic surfactant was added to reduce aggregation and denaturation. This drug had increased stability in freeze dried form and subsequent dissolution. Also, US-A-4783441 (Hoechst) discloses an aqueous solution containing a protein, such as insulin, and surfactant.EP 77870 (Green cross) discloses the addition of amino acids, monosaccharides, oligosaccharides or polyalcohols, xylytol or a hydrocarbon carboxylic acid of uluchsheniyu to aqueous solution of factor VIII to increase stability during processing by heating.WO-A-91/10439 (Octapharma) declares a stable solution of factor VIII or factor IX for injection, which contains a disaccharide, preferably, sucrose, and one or more amino acids, and EP 315968 and EP 314095 (Rorer) represent stable preparations of factor VIII of different ionic strength.US-A-4727027 (Diamond Scientific) is directed to a method of photochemical decontamination of aqueous compositions containing biologically active proteins isolated from blood or blood components, to minimize loss of activity. This method includes adding at least one furocoumarin to the composition and irradiating the resulting composition with ultraviolet light (UV). Before irradiation, the concentration of oxygen in the aqueous composition may be reduced for inhibiting denaturation. The latter is achieved, for example, by adding oxygen absorbers, albumin and/or enzyme systems and/or flushing with an inert gas. Solutions containing factor VIII was rinsed with argon with ascorbate and without ascorbate over a period of time up to 6 hours, US-A-4727027 silent about storage solutions for a long time, and also about the possible effects of reduced oxygen concentration on the activity of factor VIII in this storage.EP-A-0212040 (Measurement) otnosit oxygen. Heating was carried out in the absence of stabilizers, since the latter also protects and viruses, reducing, thus, the processing efficiency. The tests were performed at 90oC for 30 hours, EP-A-0212040 silent about the problem of low stability of aqueous solutions containing factor VIII, which is usually much more difficult problem than the low stability of dry products. This is due to chemical changes, for example, hydrolysis and deamination, which are much more pronounced in the solution than in the dry matter.Proteins differ in their physico-chemical properties. In the preparation of a pharmaceutical preparation, which must be physiologically acceptable and stable for a long time, it is impossible to take into account only the properties of the protein; must be taken into account also other aspects. Examples of the latter include industrial production, as well as ease of use and safety for patients. When testing different drugs consequences of these aspects unpredictable, and often for each protein there is only one solution.The use and production of factor VIII easier nego a long shelf life. This solution also facilitates patient use of the final drug product. The patient may be injected end of a medicinal product directly, without dilution.Aqueous solutions containing chemical compounds that are sensitive to oxygen, can be deoksigenirovanii as follows.Water for injection is blown with nitrogen to reduce the oxygen concentration. The components are dissolved and the solution is rinsed with nitrogen and stored in a nitrogen atmosphere. During filling, the bottles are rinsed with a stream of nitrogen and the bottles are closed under a stream of nitrogen. However, deoxygenate protein solution by blowing the solution gas is impossible. Protein solutions strongly foams and many protein drugs, such as coagulation factor VIII, denature when exposed to such treatment. Therefore, previously not offered storage of an aqueous solution containing factor VIII clotting under an inert gas such as nitrogen.Description of the invention
We have found that solutions containing coagulation factor VIII, can be deoksigenirovanii without denaturation of the protein. So, to our surprise, we found that the coagulation factor VIII can be stabilized without and oC. Thus, the present invention relates to the final drug product, including factor VIII in aqueous solution with low concentration of oxygen for a significant preservation of the activity of factor VIII with long-term storage.Factor VIII can be either a plasma factor VIII and recombinant factor VIII. When factor VIII is recombinant, he may be in the form of full length or, preferably, in the form of fragments. More preferably, these fragments are cleavage products (fragments) FVIII SQ (r - VIII SQ). The activity of factor VIII can be from 10 to 100 000 IU/ml, preferably, from 50 to 10 000 IU/mlFactor VIII used in the examples is vysokotsenny, i.e. has a specific activity of more than 5000 IU/mg protein, and composition according to the present invention, stabilized without the addition of albumin.The oxygen concentration can be reduced by subjecting the aqueous solution to effect the atmosphere of inert gas, or first reducing pressure, and then introducing an inert gas. The latter process is preferably repeated in several cycles. In this way the oxygen content in the solution can be reduced to neschastij per million, convenient - less than 50 ppm, preferably below 10 ppm and most preferably below 2 ppm. The oxygen content in the container can be reduced in the same way, preferably by exposure to the atmosphere of inert gas.The final medicinal product refers to the medicinal product in its final container. Suitable containers in the present invention are, for example, vials, syringes and devices for injection.A good solution is maintained under an inert gas, such as nitrogen, argon or helium, which largely supports low oxygen content. The inert gas preferably is ignoble and, most preferably, nitrogen.Low oxygen levels can largely be supported by the PCI add antioxidant to aqueous solution. So, the solution preferably contains additionally at least one antioxidant, such as glutathione, acetylcysteine, methionine, tocopherol, equivalent, butylhydroxyanisole or phenolic compounds. Preferably, the antioxidant is at least one compound selected from the group costaud further improve the stability of factor VIII.The amount of antioxidant depends on the selected connection. Consequently, it is impossible to bring the total concentration or quantity. It is important, however, that the amount of the antioxidant, if it applies; it would be a pharmaceutically acceptable amount.A suitable pH value of the solution is from 6:6 to 8.5, preferably about 7. Preferably, the solution was attended by non-ionic surface-active agent. Non-ionic surface-active agent, if used, is preferably selected from block copolymers, such as poloxamer or fatty acid ester and polyoxyethylene sorbitan, such as Polysorbate 20 or Polysorbate 80. Non-ionic surface-active agent, if applicable, must be present in an amount of more critical micelles concentration (KMK). Cm. , Wan and Lee, Journal of Pharm.Sci., 63, page 136, 1974. Thus, the fatty acid ester and polyoxyethylene sorbitan preferably be added in amounts of at least 0.01 mg/mlThe aqueous solution may additionally contain sodium chloride or potassium, preferably in amounts of more than 0.1 MThe Association of heavy and light chains of factor VIII depends on the presence of calcium or other ions dvukhvalentnymi, such as calcium gluconate, glubionate calcium or gluceptate calcium, preferably more than 0.5 mm.The aqueous solution preferably contains the amino acid, such as L-histidine, lysine and/or arginine, in the amount of more than 1 mm. You can add mono - or disaccharides, such as sucrose or polyalcohols, xylytol. Preferably, the solution contains L-histidine and sucrose.The final drug product preferably consists of an aqueous solution containing:
1) 10-100 000 IU/ml of recombinant coagulation factor VIII;
2) at least 0.01 mg/ml fatty acid ester and polyoxyethylene sorbitan;
3) sodium chloride, preferably more than 0.1 M;
4) a salt of calcium, such as calcium chloride or calcium gluconate, preferably in quantities of 0.5 mm;
5) amino acid such as L-histidine, in the amount of more than 1 mm;
6) mono - or disaccharide or sugar alcohols, preferably sucrose or mannitol.To this solution may be added an antioxidant in pharmaceutically acceptable amounts.Thus, the final drug product comprises a stable aqueous solution, ready to use.The claimed solution can b the ri which factor VIII clotting elute aqueous solution in the last step of purification. The aqueous solution preferably contains at least one additional substance selected from the group consisting of nonionic surface-active agent, antioxidant, amino acids such as L-histidine, sodium salt, calcium salt and sucrose.The present invention relates also to a method of improving the stability of coagulation factor VIII in an aqueous solution, wherein the solution is stored under the atmosphere of inert gas. The present invention relates also to the method by which it is possible to save at least 50% activity, and even 80% of the activity of factor VIII from the original, after storage for at least 6 months at a temperature of from 2 to 10oC and pH from 6.5 to 8.5. Using the present invention, it is possible to store medicinal product containing factor VIII in aqueous solution, 12 and even 24 months without significant loss of activity of factor VIII. This method is particularly suitable for the case when factor VIII is r-VIII SQ, because the data presented in the examples show that r-VIII SQ, very stable for up to at least 6 months if stored under nitrogen at 53oC.The following examples illustrate the present invention and p is m, according to the present invention, and the air for comparison. Patent protection is not limited to these examples.Experimental part
Materials and methods
The preparation of recombinant factor VIII SQ (r-VIII SQ) was done as described in patent WO-A-91/09122, example 1-3.DHFR-deficient cell line CHO (DG44N.Y) electorical expressing vector, containing the gene for r-VIII SQ, and expressing the vector containing the gene digidrofolatreduktazy. After selection on selective medium surviving colonies amplified by growth in gradually increasing amounts of methotrexate. The supernatant from the resulting colonies individually investigated on the activity of factor VIII. Was selected productive clone and adapted to suspension growth in the environment of a particular composition that does not contain serum, the result of which was developed a large-scale enzymatic process. The supernatant was collected after a certain period of time and then purified as described below.The clarified conditioned medium was given a certain pH value and was placed on a column of S-separate FF. After serving the factor VIII was suirable aleluia monoclonal antibody (A), directed against the heavy chain of factor VIII. Before loading on the column S-eluate was treated with 0.3% TNBP and 1% Octoxynol 9. The column was balanced, washed and factor VIII were suirable buffer containing 0.05 M CaCl2and 50% of ethylene glycol.mAb - eluate was loaded on a column of Q-separate FF, balanced eluting buffer on immunoaffinity stage. After laundering factor VIII was suirable 0.05 M L-histidine, 4 mm CaCl2, 0.6 M NaCl, pH 6,8.Q-eluate was placed in a gel-filtration column (Superdex 200 p.g.). The equilibration and elution was performed with buffer, according to the recipe of the drug, which received the composition, according to the following examples.The volume of material r-VIII SQ was received after the final purification steps. The activity of factor VIII and the concentration of the inactive components are brought up to the desired by diluting the appropriate buffer. The solution is then sterilized by filtration (0.22 μm), were Packed up and desoxyribose by placing the solution in conditions of reduced pressure and the subsequent introduction of inert gas several cycles.The activity of coagulation factor VIII was evaluated by chromogenic substrate assay. (Coatest Factor VIII, Chromogenis AB, Moindal, Sweden). Intense activity as a cofactor. Factor Xathen determined by using a synthetic chromogenic substrate, S-2222, in the presence of thrombin inhibitor 1-2581 to prevent hydrolysis of the substrate by thrombin. The reaction was stopped with acid, a VIII:C, which is proportional to the release of PNA (para-nitroaniline) was determined photometrically at 450 nm in comparison with the control reagent. Unit of factor VIII: C was expressed in international units (ME), as defined by the current international standard (MS) concentrations established by who.Example 1. Comparison of solutions at storage in nitrogen atmosphere or air.Recombinant factor VIII was obtained as described under "Experimental". The solutions were stored at three different temperatures: 7, 25 and 30oC, respectively. Volume of packaged drug in vials was 2 ml (table. 1)
From this example it is clear that the absence of oxygen during the 7oC gives an acceptable loss of VIII:C after 6 months when stored in solution. Even at 25 or 30oC the solution according to the present invention can be stored without too much loss of activity. Next, you can see that the stability was better at pH 7 than at pH 6.Prime "Experimental". The solutions were stored at two different temperatures, 7 and 25oC, respectively. Volume of packaged drug in vials was 2 ml (table.2).Both solutions showed acceptable stability VIII:C six months during the 7oC.Example 3. Comparison of solutions containing glutathione or ascorbic acid, and stored in an atmosphere of air or nitrogen.Recombinant factor VIII was received, as described under "Experimental". The solutions were kept at a temperature of 25oC. Volume of packaged drug in vials was 2 ml (table. 3).After 2 months of storage under nitrogen atmosphere, the activity of factor VIII was maintained at approximately 80% of the initial value or more. This was observed regardless of the presence or absence of glutathione. However, glutathione significantly increased the storage stability of the solution under atmospheric air. Ascorbic acid reduced the stability of factor VIII.Example 4. Comparison of solutions with glutathione and without glutathione, stored under argon or air
Recombinant factor VIII was obtained as described in the section."Experimental castle in the vial was 2 ml (table. 4).After 6 months of storage under argon, the activity of factor VIII was maintained at approximately 65% or more from the initial value at a storage temperature of 25oC. After storage at a temperature of 7oC the value was 80% or more. 1. A stable aqueous solution of coagulation factor VIII, characterized in that it includes coagulation factor VIII activity from 10 to 100,000 IU/ml in aqueous solution with reduced oxygen concentration.2. A stable aqueous solution of coagulation factor VIII under item 1, characterized in that it additionally contains an inert gas.3. A stable aqueous solution of coagulation factor VIII under item 1, characterized in that as the inert gas using nitrogen.4. A stable aqueous solution of coagulation factor VIII in PP.1 to 3, characterized in that the aqueous solution additionally contains at least one antioxidant, such as glutathione, acetylcysteine, methionine, tocopherol, equivalent, butylhydroxyanisole or phenolic compounds.5. A stable aqueous solution of coagulation factor VIII under item 4, characterized in that the antioxidant is of ethionine.6. A stable aqueous solution of coagulation factor VIII according to any one of paragraphs. 1 to 5, characterized in that as coagulation factor VIII using highly purified coagulation factor VIII, and the solution is stable without the addition of albumin.7. A stable aqueous solution of coagulation factor VIII according to any one of paragraphs. 1 to 5, characterized in that as coagulation factor VIII using coagulation factor full length or a fragment of recombinant factor VIII.8. A stable aqueous solution of coagulation factor VIII according to any one of paragraphs.1 to 7, characterized in that use active coagulation factor VIII in a concentration of from 50 to 10,000 IU/ml9. A stable aqueous solution of coagulation factor VIII according to any one of paragraphs.1 to 8, characterized in that use an aqueous solution, optionally containing a non-ionic surface-active agent.10. A stable aqueous solution of coagulation factor VIII under item 9, characterized in that use non-ionic surface-active agent in amounts exceeding the critical concentration of micelle formation.11. A stable aqueous solution of coagulation factor VIII according to any one of p is OSTO-active agent, selected from block copolymers or fatty acid ester and polyoxyethylene sorbitan.12. A stable aqueous solution of coagulation factor VIII according to any one of paragraphs.1 - 11, characterized in that use an aqueous solution, optionally containing a salt of calcium, such as calcium chloride or calcium gluconate, preferably in excess of 0.5 mm.13. A stable aqueous solution of coagulation factor VIII according to any one of paragraphs.1 - 12, characterized in that use an aqueous solution, optionally containing amino acid such as L - histidine, in excess of 1 mm.14. A stable aqueous solution of the coagulation factor XIII according to any one of paragraphs.1 - 13, characterized in that use an aqueous solution, optionally containing mono - or disaccharides or polyalcohols, xylytol, preferably sucrose.15. A stable aqueous solution of coagulation factor VIII according to any one of paragraphs. 1 to 14, characterized in that use an aqueous solution containing 10-100.000 IU/ml of recombinant coagulation factor VIII, at least 0.01 mg/ml fatty acid ester and polyoxyethylene sorbitane, sodium chloride, preferably in excess of 0.1 M, a salt of calcium, such as chloride ka is in the number, exceeding 1 mm, mono - or disaccharide or sugar alcohols, preferably sucrose or mannitol.16. The method of obtaining a stable aqueous solution of the coagulation factor VIII according to any one of paragraphs.1 - 15, characterized in that the coagulation factor VIII is mixed with an aqueous solution to obtain a solution with an activity of from 10 to 100,000 IU/ml and lower concentrations of oxygen by exposure to a solution of inert atmospheric gas.17. The method of obtaining a stable aqueous solution under item 16, characterized in that the coagulation factor VIII is mixed with an aqueous solution and reduce the oxygen concentration by reducing the pressure and the subsequent introduction of inert gas, preferably repeated in several cycles.18. The method of obtaining a stable aqueous solution according to any one of paragraphs.16 and 17, characterized in that the coagulation factor VIII elute at the last stage aqueous buffer solution and reduce the oxygen concentration by treatment of the solution with inert gas.19. The method of obtaining a stable aqueous solution according to any one of paragraphs.16 to 18, characterized in that the coagulation factor VIII elute at the last stage water purification buffer rappocciolo repeated in several cycles.20. The method of obtaining a stable aqueous solution according to any one of paragraphs.16 to 19, characterized in that the aqueous solution contains at least one additional substance selected from the group consisting of nonionic surface-active agent, antioxidant, amino acids such as L - histidine, sodium salt, calcium salt and sucrose.
FIELD: medicine, ophthalmology.
SUBSTANCE: one should apply an autohemocomponent preparation being supernatant liquid of patient's autoblood at increased serotonin content obtained due to irreversible thrombocytic aggregation due to the impact of 0.5 mg ATP per 1.0 ml plasma followed by a 30-min-long centrifuging at the rate of 1000, 2000 and 3000 rot./min for 20, 7 and 3 min, correspondingly. In case of no exudative phenomena on patient's eye bottom the obtained preparation should introduced at the quantity of 7-10 ml once in 48 h for 1 mo (totally, 15 injections). In case of exudative-hemorrhagic phenomena it should be introduced parabulbarly at the volume of 0.5 ml and parenterally - 7.0-10.0 ml once in 48 h for 1 mo per 15 injections, correspondingly. The preparation enables to improve visual functions due to decreased tissue hypoxia and normalization of microcirculation in visual analyzer.
EFFECT: higher efficiency of therapy.
2 cl, 7 dwg, 2 ex
FIELD: medicine, ophthalmology.
SUBSTANCE: one should apply an autohemocomponent preparation being supernatant liquid of patient's autoblood at increased serotonin content obtained due to irreversible thrombocytic aggregation due to simultaneous impact of 0.5 mg ATP per 1.0 ml plasma followed by a 30-min-long centrifuging at the rate of 1000, 2000 and 3000 rot./min for 20, 7 and 3 min, correspondingly. Preparation should introduced parabulbarly at the volume of 0.5-1.0 ml, parenterally - 7.0-10.0 ml once daily at 20 injections for 1 mo. The method enables to increase visual functions due to normalization of functional activity of photoreceptors and neurons of internal layer in retinal peripheral departments.
EFFECT: higher efficiency of therapy.
1 cl, 2 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
FIELD: biotechnology, medicine, pharmacy, veterinary science.
SUBSTANCE: method involves addition of DEAE-Sephadex A-50 to cryosupernatant from human blood plasma, incubation, filtration and addition of QAE-Sephadex to filtrate followed by incubation. Filtered off precipitate of QAE-Sephadex is subjected for successive step-by-step washing out with buffer solution at pH 5.5 and 7.5, elution at pH 7.7 and dialysis. Then PEG-6000 is added to dialyzed solution to the concentration 12%, solution is incubated and centrifuged. To the prepared supernatant glycine is added to the final concentration 100 mM and lysine is added to the final concentration 10 mM at pH 7.2, then Twin-80 is added and pH value is corrected to 6.8-7.2 followed by addition of tri-n-butyl phosphate to the final concentration 0.3%. Prepared suspension is stirred, subjected for chromatography on DEAE-Sepharose FF at pH 7.0, chromatography on Zn-chelating Sepharose FF at pH 7.5 and the end product with specific activity from 7.5 ± 0.5 U/mg of protein and above, and with the final concentration of lysine 10 mM, not less, and with the final concentration of glycine 100 mM, not less. Method provides safety of activity in antiviral treatment and preparing product containing the natural C-1 esterase inhibitor from blood plasma with high specific activity.
EFFECT: improved method for preparing.
6 cl, 2 dwg, 1 ex
FIELD: medicine, ophthalmology.
SUBSTANCE: beforehand one should perform parenteral antibioticotherapy followed by close vitrectomy followed by filling an eye with autoserum. The method provides suppression of bacterial flora intraocularly, decrease toxic impact upon ocular structures and retinal function with substitutors of vitreous body and antibiotics.
EFFECT: higher efficiency of therapy.
FIELD: medicine, biotechnology, pharmaceutical industry, veterinary science.
SUBSTANCE: method involves addition of tris-HCl buffer (pH 7.5) containing 0.1 M of lysine and 5 mM of EDTA to the Cohn's fraction followed by incubation and centrifugation. Prepared suspension is incubated with 10% PEG-3000 and after centrifugation the solution is applied onto column with lysine-Sepharose at pH 7.5 and plasminogen is eluted with buffer solution at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine and 5 mM of caprylic acid. Then plasminogen is incubated with streptokinase in tris-buffer (pH 7.5) containing 15 mM of lysine, 10 mM of ε-aminocaproic acid and 50% of glycerol. Then method involves chromatography on column with benzamidine-Sepharose at pH 8.5 and elution with buffer at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine, and the end product is lyophilized that comprises about 30 IU/ml of plasmin with purity above 98%, at least 10 mM of lysine and 10 mM of glycine in an aqueous solution at pH about 3.5. The prepared product shows apyrogenic property, absence of toxicity in experiments in laboratory animals and it doesn't cause allergic or other adverse response reactions in intracutaneous or intravenous administrations, and the preparation doesn't cause defects in the vision field after its an intraorbital route of administration.
EFFECT: improved preparing method, valuable medicinal properties of plasmin.
7 cl, 1 ex
FIELD: medicine, oncourology.
SUBSTANCE: the present innovation deals with conservative treatment of patients with malignant prostatic tumor at different stages. The method includes testicular enucleation, introduction of anti-tumor chemopreparations and radiation therapy. Moreover, in the onset of radiation therapy one should introduce 25 mg Cisplatin incubated with 10 ml patient's plasma into both prostatic lobes and paraprostatic fiber from the right and from the left. At achieving a focal dosage of 20 Gy one should repeat introduction of chemopreparation in similar dosage, and radiation therapy should be continued up to total focal dosage of 40 Gy. The innovation enables to decrease tumor sizes, side manifestations of radiation therapy at decreasing radiation loading and improve patient's life quality due to mitigating the urination.
EFFECT: higher efficiency of therapy.
FIELD: medicine, oncology.
SUBSTANCE: after removing malignant cerebral tumor one should conduct successive course of: cytokinotherapy consisting of 3 intramuscular injections of leukineferon at 48-h-long interval, adaptive immunotherapy with lymphokine-activated killer cells (LAKC) generated in the presence of recombinant interleukin-2 (IL-2). Moreover, LAKC should be introduced into the channel of removed tumor in combination with IL-2 as 2 procedures at 24-h-long interval. Then comes the course of adaptive immunotherapy with cytotoxic lymphocytes (CTL) generated due to cultivating patient's mononuclear blood cells together with dendritic cells loaded with a tumor antigen, in the presence of recombinant IL-2 to be introduced in combination with it into the channel of removed tumor as 2 procedures at 48-h-long interval. For obtaining dendritic cells in patients before operation it is necessary to isolate monocytes to cultivate with granulocytic-macrophageal colony-stimulating factor and interferon-α at maturating dendritic cells in the presence of monocytic conditioned medium and incubation of dendritic cells in the presence of tumor antigenic material for their loading with a tumor antigen. After immunotherapy with CTL on should conduct the course of vaccinotherapy with dendritic cells loaded with a tumor antigen in combination with subcutaneous injections of IL-2. The method enables to induce high specific anti-tumor immune response along with improved quality of life and prolonged duration of relapse-free period.
EFFECT: higher efficiency of immunotherapy.
2 cl, 2 ex
FIELD: medicine, obstetrics.
SUBSTANCE: the present innovation deals with introducing the complex of medicinal preparations directed for treating different complications during pregnancy. Moreover, along with applying the above-mentioned complex it is necessary to introduce actovegin as uterotonic remedy per 2 tablets thrice daily. The innovation enables to normalize myometrium state and that of uterine-placental circulation at efficiency being 93%.
EFFECT: higher efficiency.