The composition containing the product of coagulation factor viii, its preparation and application of surfactants as stabilizer
(57) Abstract:The invention relates to medicine and can be used to treat patients suffering from hemophilia. A pharmaceutical composition comprising a recombinant coagulation factor VIII, as a stabilizer for long term storage is Polysorbate 80, sodium chloride or potassium, a salt of calcium and L-histidine to tebufelone system. The method of obtaining the claimed compositions by blending factor VIII with Polysorbate 80 and other components or by chromatographic purification of recombinant factor VIII and elution of it at the last stage of purification buffer containing Polysorbate 80. The use of Polysorbate 80 as a stabilizer compositions containing recombinant coagulation factor VIII. The invention provides stable during storage of the pharmaceutical composition without the addition of albumin. 4 C. and 18 h.p. f-crystals, 2 Il. The invention relates to new compositions containing coagulation factor VIII and nonionic surfactant, such as a block copolymer, for example, poloxamer, or ester polyoxyethylenated (20) the derivative of sorbitol with a fatty acid, in particular Polysorbate 20 or Polysorbate 80. Comp">Hemophilia is a congenital disease that has been known for many centuries, but only in the last three decades has managed to differentiate its varieties: hemophilia A, hemophilia B and hemophilia C. Haemophilia A is the most common form. This disease affects only men with a frequency of one or two individuals per 10,000 men. The disease is a consequence of the greatly reduced levels or absence of biologically active coagulation factor VIII (antihemophilic factor), a protein that is usually found in the plasma. Clinical manifestation of hemophilia is expressed in a strong tendency to bleeding, and before the introduction in practice of treatment with the use of concentrates of factor VIII the average life expectancy of these patients were less than 20 years. Concentrates of factor VIII obtained from plasma available for over three decades. This has significantly improved the ability to treat patients suffering from hemophilia, and allowed them to lead a normal life.Therapeutic concentrates of factor VIII, until recently, were prepared by plasma fractionation. Currently, however, developed new ways of producing factor VIII in the cells is J. Gitschier et al., Nature 312, 330-337, 1984 and in the European patent 160457.Concentrates of factor VIII isolated from human plasma, contain several fragmentary fully active forms of factor VIII (Anderson et al., Proc. Nat. Acad. Sci, USA, Vol. 83, 2973-83, May 1986). The smallest active form has a molecular mass of 170 kilodaltons and consists of two chains with a mass of 90 kilodaltons and 80 kilodaltons, which are connected to each other by a bridge made of metal ion (see European patent 197901). Firm "Kabi Pharmacia has developed derived recombinant factor VIII, the corresponding form factor VIII from plasma, which has a mass of 170 kilodaltons in therapeutic concentrates of factor VIII. Molecules of a truncated factor VIII is called r-VIII SQ and produced as the end product of the ovary cells of Chinese hamsters in processes using cell cultures.The specific activity of r-VIII SQ may be more than 12000 M. E./mg protein and is preferably more than 14000 M. E./mg was Observed activity of about 15000 M. E./mg. Activity approximately 10,000 M. E. VIII:C per mg protein was previously shown to our r-VIII SQ.Recombinant factor VIII SQ is recommended for the treatment of classical hemophilia. Dosage similar dose conreport, for injection is required only small amounts.General information about the structure and biochemistry of recombinant derivatives of factor VIII described by Kaufman in Tibtech., Vol. 9, 1991 and Hamatology, Vol. 63, 155-65, 1991. Structure and biochemistry of r-VIII SQ is described in International patent WO 91/09122.The stability of proteins is a common problem in the pharmaceutical industry. It is often solved by drying the protein using various drying methods such as freeze drying. After that, proteins are Packed up and stored in dry form.Solution before drying or freeze-drying, the dry matter and the newly restored from it the product must be stable, so that not too much activity was lost in the drying process, during storage and use.Factor VIII, highlighted by the fractionation of the plasma, usually sold in the form lyophilizers powder, which before use is necessary to dilute with water.The compounds with a small amount of protein in General lose activity during purification, sterilization, packaging while taking medication. This problem is usually solved by the introduction of albumin human, which significantly and cleaning, sterilization in the production process and freeze-drying (see review by Wang et al., J. of Parenteral Sci. and Tech. Vol. 42, No. 42, Supplement 1988). Albumin person also contributes to the formation of the pie when the freeze-drying process. The use of albumin to stabilize factor VIII is known and is currently used in all coming to market highly pure derivatives of factor VIII.However, the addition of albumin in therapeutic protein produced by recombinant DNA technology is undesirable. The use of albumin as an excipient often limits the ability of many of the most powerful and sensitive analytical techniques used to study proteins.You must get free from albumin compositions containing factor VIII, in particular recombinant factor VIII, which would be sustainable when conducting drying or freeze-drying, stable in solution or in the form of a solution after preparation of the preparation of the powder.For the stabilization of the different proteins was proposed several solutions:
In the European patent 35204 (Cutter) describes the methods that provide thermal stability of the protein composition in prists the presence carboximetilcelulosa.In International patent publication WO 89/09614 (Genentech) describes a stabilised composition comprising human growth hormone, which contains glycine, mannitol and the buffer, and in the preferred embodiment of the invention also nonionic surfactant, such as Polysorbate 80. Nonionic surfactant is introduced to reduce aggregation and denaturation. The composition has a high stability in the form of liofilizovannyh songs and after their dissolution in the media.In the European patent 268110 (Cetus) applying a solution comprising a specific protein interleukin-2, which is dissolved in inert media, including as a solubilizer/stabilizer nonionic polymeric surfactant. The preferred detergents are polyoxyethylene octylphenoxypoly ethanol derivatives of the monostearate of polyethylene glycol and esters of polyethylenimine and fatty acids.In U.S. Patent 4783441 (Hoechst) is an aqueous solution comprising a protein, such as insulin, and surfactant.In U.S. Patent 4165370 (Coval) describes the solution of gammaglobulin and the way his prigot the society.In the European patent 77870 (Gren Cross) claimed a method of increasing the stability of solutions of factor VIII, which consists in adding amino acids, monosaccharides, oligosaccharides or sugar alcohols or carboxylic acids, and the addition of alcohols, sugars or disaccharides to aqueous solutions of factor VIII to increase stability in the heat treatments described in the European patent 117064 (Green Cross).In the International patent WO 91/10439 (Octopharma) declared stable solution for injection of factor VIII or factor IX, which contains a disaccharide, mainly sucrose, and one or more amino acids.In the European patent 315968 and European patent 314095 (Rorer) are reported as stable compositions of different ionic strength, containing factor VIII.Proteins differ in their physico-chemical properties. Upon receipt of pharmaceutical preparations, which must be physico-chemically acceptable and sustainable for a long time, consider not only the physiological properties of the protein, but also other aspects such as the possibility of industrial production, ease of use by the patient and safety for the patient. Implications of su is th protein is the only possible solution.Factor VIII contained in the plasma is stabilized by Association with protein carrier-factor von Willebrand's disease. In plasma, as well as in conventional concentrates of factor VIII intermediate purity ratio factor a background of Villebranda to factor VIII is at least 50:1 by weight. In very pure concentrates of factor VIII has a specific activity greater than 2000 m is mg protein, the factor a background of Villebranda to factor VIII is approximately 1:1 (by weight) and basically the whole factor VIII attached to the factor a background of Villebranda. Despite this stabilization to achieve acceptable stability during lyophilization and storage requires additional stabilization due to the introduction of albumin.All too pure drugs, marketed, stable albumin (albumin human serum).Currently requires factor VIII for injection without albumin, containing the minimum amount of additives.We have developed new compositions which allow us to resolve these problems for factor VIII.Unexpectedly, it was found that factor VIII, which is a very sensitive protein may be stabilized without albumin to the composition, includes coagulating factor VIII and nonionic surfactant as a stabilizer. Our factor VIII is a highly pure, i.e., has a specific activity of more than 5,000 IU/mg protein, and the composition is stable without the addition of albumin.If factor VIII is recombinant, it can be either full-size or deletion derivative, such as a derivative SQ.The number of factor VIII is from 10 to 100,000 IU/ml, mostly from 50 to 10,000 IU/mlNonionic surfactant predominantly selected from a block copolymer, such as poloxamer, or a complex ester of polyoxyethylene (20) and fatty acids, such as Polysorbate 20 or Polysorbate 80. As Polysorbate 80 is used Tween 80.Nonionic surfactant should be present in an amount above the critical micellization concentration (CMC) (see Lee, Journal of Pharm. Sci., Vol. 63, 136, 1974).Ester of polyoxyethylene (20) and fatty acids mainly contains, therefore, in the amount of at least 0.01 mg/ml, It can be, in particular from 0.02 to 1 mg/ml of the Composition may also include sodium chloride or Kali is such as calcium chloride or calcium gluconate, preferably not less than 0.5 mm, and an amino acid such as L-histidine in an amount of not less than 1 mm. This quantity can be selected in the range from 0.05 to 500 mm.Can be added mono - and disaccharides, such as sucrose or sugar alcohols, in particular in an amount of from 1 to 300 mg/ml.Composition mainly contains L-histidine and sucrose. The ratio in the composition of sodium chloride to L-histidine is at least 1:1.The composition may include:
i) 10-100000 M. E./ml of recombinant factor VIII;
ii) at least 0.01 mg/ml of ester of polyoxyethylene (20) and fatty acids;
iii) sodium chloride, predominantly in the amount of not less than 0.1 M;
iv) a salt of calcium, such as calcium chloride or calcium gluconate, mainly in the amount of not less than 0.5 mm;
v) an amino acid such as L-histidine in an amount of not less than 1 mm.To this composition mainly added mono - or disaccharides or sugar alcohols.The composition may be in solid form, mainly liofilizovannyh, or in the form of an aqueous solution before or after drying. The dried product is diluted with sterile water for injection or buffer solution.The inventive composition can be obtained by blending factor VIII with nonionic surface-active agent in an aqueous solution, mainly together with amino acid such as L-histidine, sodium salt, sucrose and calcium salt or by elution of factor VIII in the last step of purification buffer containing aqueous solution of nonionic surfactant, predominantly with amino acid such as L-histidine, sodium salt, sucrose and calcium salt.The invention relates also to the use of a stabilizer composition comprising coagulation factor VIII, nonionic surfactants, which are predominantly selected from a block copolymer, preferably poloxamer or a complex ester of polyoxyethylene (20) and a fatty acid, preferably Polysorbate 20 or Polysorbate 80.The amino acid used as a buffer, and to protect the protein in the amorphous phase. A suitable buffer can serve as L-histidine, lysine and/or arginine. Mainly use L-histidine, pascalcase sucrose or sugar alcohol.The calcium (or ions of divalent metal), in this case in the form of calcium chloride, although it may be added, and other salts such as calcium gluconate, glubionate calcium or gluceptate of calcium, necessary for binding the larger and the smaller chains of factor VIII.Presented in examples data show that r-VIII SQ when stored at a temperature of 53oC stable for at least 12 months.The following examples explain the invention and contain data on the stability of different formulations, each of which falls under the scope of the claims of the invention and are not limited to these examples.In Fig. 1 shows the data obtained by the method of gel permeation chromatography using liquid chromatography high pressure, obtained for example 10A after storage for 5 months at a temperature of 25oC.In Fig. 2 - data obtained by the method of gel permeation chromatography using liquid chromatography high pressure, obtained for example 10B after storage for 5 months at a temperature of 30oC.The preparation of recombinant factor VIII SQ (r-VIII SQ) is carried out mainly according to the method, the PRIOME electropolishing with the expression vector, containing the gene for r-VIII SQ, and the expression vector containing the gene of dihydrofolate-reductase. By gradually increasing the content of methotrexate pursue a coherent selection of surviving colonies of the selected environment. The liquid above the precipitate obtained colonies subjected to individual screening VIII:C activity. Choose the clone for production, which is then ready for growth in not containing serum suspension in the selected environment, and finally develop the full process of obtaining. The liquid above the precipitate is taken after a certain period of time and subjected to further treatment, as described next.Install the necessary pH clarified and brought to the condition of the environment and transfer it into a column filled with S-Sepharose FF. After washing, factor VIII elute salt buffer solution containing 5 mm calcium chloride.The study of immunoadsorption spend on immunoaffinity resin, a ligand which is a monoclonal antibody (8A4). acting on a long chain of factor VIII. Before loading to the column S-eluate was processed with a solution containing 0.3% TNBP and 1% octoxynol 9.Column lead in equilibrium, washed the at is placed in a column, filled with Q-Sepharose FF, and when conducting immunopheno analysis result in the equilibrium state of the elution buffer solution.After washing, factor VIII elute 0.05 M solution of L-histidine, 4 mm calcium chloride and 0.6 mm solution of sodium chloride with a pH of 6.8.About the eluate was placed in a column for filtration (Superdex 200 p.g.). Bringing in the equilibrium state and the elution is performed using a composition containing sodium chloride, L-histidine, calcium chloride and Polysorbate 80.Collect the fractions containing the protein, and prepare a solution before freeze drying.Set the activity VIII: C and the concentration of active components by dilution with a suitable buffer. The solution is then sterilized by filtration (0.22 μm), spray and subjected to freeze-drying. Samples of each composition is frozen and stored at -70oC. These samples after thawing used as a standard in research VIII:C.The coagulating ability of VIII: C is determined according to the method pigmentbased substrate (Coatest Factor VIII, "Chrovogenix AB, Molndal, Sweden). Activated factor X (Xa) is generated on the internal mechanism, and the factor VIII:C plays a role prisutstvie thrombin inhibitor 1-2581 to prevent hydrolysis of the substrate by the action of thrombin. The reaction is interrupted by the addition of acid, and the content of VIII: C, which is proportional to the allocation of p-nitroaniline, determined photometrically at a wavelength of 450 nm with respect to pure reagent. The content of factor VIII: C is expressed in international units (M. E.) in accordance with the requirements of the International standard for concentrates, adopted by the world health organization.Regeneration VIII:C is calculated in percent as the ratio of the content of VIII:C in the newly prepared solution divided by the contents VIII:C in frozen, and then Ottana solution for the process of freeze-drying, with appropriate adaptations for dilution.Soluble aggregate is determined by using gel permeation chromatography. Use full column Superdex 200 HR 13/30 (company "Pharmacia") and fluorescence detector (excitation frequency 280 nm band emission 340 nm). Analyze the newly prepared compounds. The evaluation of the results of gel permeation chromatography conduct a visual study of chromatograms or by integration of peak areas in the case when the formation of the aggregate.The amount of regeneration in the process of freeze drying is expressed as a percentage of Ernesto-active substances
Recombinant factor VIII receive as described in the Experimental part.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 5 ml of sterile water for injection.The compositions are presented in table. 1
The above example shows that there is no difference in the recovery of factor VIII : C when using nonionic surfactants or albumin.Example 2. The results depend on the strength of nonionic surfactants.Recombinant factor VIII receive as described in the Experimental part.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 2 ml of sterile water for injection.The composition presented in table. 2.It is easy to see that a surprisingly high stabilizing effect of the addition of nonionic surfactants.Example 3. Changes in the concentration of surfactants
Recombinant factor VIII receive as described in the Experimental part.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 5 ml of sterile in the m example show that regeneration of factor VIII (VII : C) was very high after re-dissolution and good for all used concentrations of Polysorbate 80.Example 4. Changes in the concentration of sodium chloride.Recombinant factor VIII receive as described in the Experimental part.2 ml of the solution is subjected to freeze-drying, stored at different temperatures for up to 6 months, and then re-dissolved in 5 ml of sterile water for injection.The compositions are presented in table. 4.Solutions containing 0.3 or 0.6 M sodium chloride show a very good stability. Both composition is stable when stored at 37oC for 6 months.Example 5. Changes in the concentration of L-histidine
Recombinant factor VIII receive as described in the Experimental part.2.2 ml of the solution is subjected to freeze-drying, stored at different temperatures for up to 3 months, and then re-dissolved in 5 ml of sterile water for injection.The compositions are presented in table. 5.This example shows that different amounts of L-histidine does not affect stability.Example 6. Recombinant/thawed 1, 5 and 10 times and get the values for regeneration, are presented in table. 7.The results show that the VIII : C stable after repeated cycles of freezing/thawing and that PEG-4000, which is, plays a role of protection when cooling is not required in these compositions.Example 7. The change in pH.Recombinant factor VIII receive as described in the Experimental part.2.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 5 ml of sterile water for injection.The compositions are presented in table. 8.The above example shows that pH has no significant impact in the interval from 6.0 to about 7.5.Example 8. The addition of sucrose.Recombinant factor VIII receive as described in the Experimental part.2.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 5 ml of sterile water for injection.The compositions are presented in table. 9.Sucrose is added to the solution B after the stage finish cleaning before freeze-drying.Recovery after freeze-drying is 76% for A and 87% for B. Ah.This study shows that the addition of sucrose favours regeneration VIII : C after freeze-drying.Example 9. Changes in the concentration of calcium salts.Recombinant factor VIII receive as described in the Experimental part.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 5 ml of sterile water for injection.The compositions are presented in table. 10.The presented data show that calcium chloride can be replaced by calcium gluconate.Example 10. Recombinant factor VIII receive as described in the Experimental part.2.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 5 ml of sterile water for injection. Contents VIII : C in the bubble in the newly prepared the drug is about 1000 I. E. (see tab. 11).Regeneration is good and if part of the L-histidine is replaced by sucrose.These compositions are examined by gel permeation chromatography after 5 months of storage at a temperature of 25oC and 30oC, respectively, and the results obtained are shown in Fig. 1 and 2. The only observed peaks of the C which should be observed for values smaller 40. It is evident from Fig. 1 shows that for 10A after 5 months of storage the aggregate are not detected. In Fig.2 DL 10V after 5 months of storage at 30oC there is a small amount of aggregate, the content of which is less than 2%.Example 11. Recombinant factor VIII receive as described in the Experimental part.2.2 ml of the solution is subjected to freeze-drying and then re-dissolved in 5 ml of sterile water for injection. Contents VIII : C in the bubble in the newly prepared the drug is about 500 I. E. (see tab. 12).Both composition show good stability.These compounds were studied using gel permeation chromatography and the obtained results similar to those shown in Fig. 1 and 2.Aggregirovanie not observed during storage of the composition for 5 months at 25oC and 30oC, respectively.Example 11. Recombinant factor VIII receive as described in the Experimental part.2 ml of the solution is subjected to freeze-drying, stored at different temperatures for up to 3 months, and then re-dissolved in 4 ml of sterile water for injection. Contents VIII : C in the bubble in the newly prepared drug from the group of five months at a temperature of 7oC.Example 12. Recombinant factor VIII receive according to the method described in the Experimental part.2 ml of the solution is subjected to freeze-drying, stored at different temperatures up to 3 months, and then re-dissolved in 4 ml of sterile water for injection. Contents VIII : C in the bubble in the prepared solution is about 500 I. E. (see tab. 14).Satisfactory stability is observed after storage at 7oC for 5 months. 1. A pharmaceutical composition comprising a recombinant coagulation factor VIII and the regulator of the activity of factor VIII during storage over an extended period of time, and this song is either original and of course aqueous solution, ready for use, or the original aqueous solution, which is then dried and at the end of recreate in the form of an aqueous solution before use, wherein the composition comprises a recombinant coagulation factor VIII specific activity greater than 2000 IU/mg protein and in the number of 10-100000 IU/ml, as a stabilizer is Polysorbate 80 in an amount of at least 0.01 mg/ml, sodium chloride or potassium in an amount of more than 0.1 M is a salt of calcium, such the project system and protection of the protein in the amorphous phase.2. The composition according to p. 1, wherein the factor VIII is of high purity and stable without the addition of albumin.3. Composition under item 1 or 2, wherein the factor VIII is full-length or deletion derivative of recombinant factor VIII.4. Composition according to any one of paragraphs. 1-3, characterized in that the content of factor VIII is 10-100000 IU/ml, preferably 50-10000 IU/ml5. Composition according to any one of paragraphs. 1-4, wherein the Polysorbate 80 is present in an amount above the critical concentration of micelle formation.6. Composition according to any one of paragraphs. 1-5, characterized in that it contains Polysorbate 80.7. The composition according to p. 6, wherein the Polysorbate 80 is present in an amount of not less than 0.01 mg/ml8. Composition according to any one of paragraphs. 1-7, characterized in that it contains sodium chloride or potassium, mainly in the amount of more than 0.1 M9. Composition according to any one of paragraphs. 1-8, characterized in that it contains a salt of calcium, such as calcium chloride or calcium gluconate, mainly in the amount of more than 0.5 mm.10. Composition according to any one of paragraphs. 1-9, characterized in that it contains amino acid, such as L-histidine, in the amount of b is property sucrose or sugar alcohols.12. Composition according to any one of paragraphs. 10 and 11, characterized in that it contains L-histidine and sucrose.13. The composition according to PP. 8 and 10, characterized in that the ratio of sodium chloride to L-histidine is more than 1:1.14. Composition according to any one of paragraphs. 1-13, characterized in that it includes:
10-100000 IU/ml recombinant factor VIII; at least 0.01 mg/ml of Polysorbate 80; sodium chloride, predominantly in the amount of more than 0.1 M; a salt of calcium, such as calcium chloride or calcium gluconate, mainly in the amount of more than 0.5 mm; amino acid such as L-histidine, in the amount of more than 1 mm.15. Composition according to any one of paragraphs. 1-14, characterized in that it dried.16. Composition according to any one of paragraphs. 1-14, characterized in that it is subjected to lyophilization.17. Composition according to any one of paragraphs. 1-14, characterized in that it is a stable aqueous solution, ready to use.18. Composition according to any one of paragraphs. 3-17, characterized in that the specific activity of r-VIII SQ is more than 12000 IU/mg, mostly more than 14,000 IU/mg.19. The composition according to p. 1, characterized in that the specific activity of factor VIII is more than 5000 IU/mg protein.20. The method of obtaining the composition is the first solution further comprises sodium chloride or potassium, salt of calcium, such as calcium chloride or calcium gluconate and L-histidine.21. The method of obtaining the composition of p. 1, characterized in that it includes the purification of the recombinant factor VIII obtained by fermentation, in chromatographic sequence containing at least two phases selected from the stages cation exchange chromatography, anion-exchange stages chromatography stages immunoaffinity chromatography and stages gel filtration, with subsequent elution of factor VIII from the last stage of purification using a buffer containing Polysorbate 80 in an aqueous solution, and this solution further comprises sodium chloride or potassium, a salt of calcium, such as calcium chloride or calcium gluconate and L-histidine.22. The use of Polysorbate 80 as a stabilizer compositions containing recombinant coagulation factor VIII.Priority points:
02.10.92 on PP.1-9, 11-13, 15-17, 19-21;
01.10.93 on PP. 10, 14, 18, 22;
11.06.93 under item 18.
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.
FIELD: pharmaceuticals industry, in particular new method for production of alpha-1-antitrypsin and pharmaceutical product containing the same.
SUBSTANCE: alpha-1-antitrypsin is isolated from Cohn fraction IV-1 by solubilization. Then protein admixtures and eventual viral particles are removed by polyethylene glycol, target protein is precipitated with zinc salt, viral inactivation using solvent/detergent is carried out, product is fractionated using Q-sepharose, and non-active alpha-1-antitrypsin is removed with S-sepharose to produced target product. Said product represents concentrate of human serum active alpha-1-antitrypsin having purity more than 98 % and specific activity not less than 40 IU/mg in 0.15 M sodium chloride solution.
EFFECT: increased yield of high pure active alpha-1-antitrypsin.