Direct thrombin inhibitor, possessing antiproliferative action

FIELD: medicine.

SUBSTANCE: substance of polypeptide nature with molecular weight 14350 Da, with N-end amino acid sequence, homologous phospholipase A2 of snake venom, and possessing properties of direct thrombin inhibitor of mixed type as well as antiproliferative action is separated of cobra venom Naja haje by three-stage liquid chromatography.

EFFECT: invention enables to produce selective direct thrombin inhibitor, possessing antiproliferative action.

1 tbl, 3 dwg, 10 ex

 

The invention relates to the field of biochemistry, in particular to a direct thrombin inhibitor, and can be used both in the laboratory and to obtain drugs for the treatment of diseases associated with hyperactively thrombin and/or hyperproliferative.

Thrombin (EC 3.4.21.5), serine protease is a key enzyme of the blood coagulation system, which converts fibrinogen into fibrin - based clot, participates in the regulation of many physiological and pathophysiological processes such as coagulation and anticoagulation mechanisms, thrombosis and fibrinolysis, regulation of vascular tone, and in the regulation of body development, as well as in the processes of inflammation, tissue repair, atherogenesis, carcinogenesis and development of Alzheimer's disease.

Untimely activation of blood coagulation in the blood stream can lead to sudden cardiac arrest, acute coronary syndromes (myocardial infarction and other), pulmonary peripheral vascular and stroke; venous channel that can lead to deep vein thrombosis and pulmonary embolism. The hyperproduction of active thrombin is also leading pathogenetic link syndrome intravascular coagulation. Since both external and internal path of blood clotting have a common final stage - activation of thrombin, as causing the formation of a fibrin clot and platelet thrombus, inhibition of thrombin is very logical in the treatment or prevention of such conditions.

Drugs antiterminator actions are classified as direct thrombin inhibitors (PETE) (acting directly on the molecule of thrombin - hirudin and its recombinant derivatives, as well as argatroban, melagatran), indirect thrombin inhibitors (heparin, fondaparinux), inhibitors of thrombin generation (inhibitor of factor XA), a combined group of recombinant analogues of endogenous anticoagulants (antithrombin, activated protein C cofactor II heparin). Existing data show that PETE have for a number of reasons, the advantage over other thrombin inhibitors in the treatment of these conditions as well as during cardiac interventions.

However, the effective application of the developed PIT is often limited to their specific disadvantages. So, hirudin and its recombinante analogues have a short half-life and side effects such as bleeding and anaphylactic reactions; most of the shortcomings of their lack Hirulog-1, a synthetic analog of hirudin, but he is easily hydrolyzed by thrombin. Melagatran (in the form of poleca the STW ximelagatran), low-molecular PETE, is a highly active inhibitor of thrombin and almost devoid of most of the shortcomings of the other PIT, but it turned out that he has hepatotoxic effects [A. Schwienhorst Direct thrombin inhibitors - a survey of recent developments // Cell Mol. Life Sci. - 2006. - V.63. - P.2773-2791].

Despite huge selection of low-molecular-weight effectors of the blood coagulation system, in order to thin the biochemical characteristics of hemostasis widely used drugs and diagnostics based on proteins from venoms of snakes. In particular, in standard laboratory practice to determine whether pathological inhibitors of polymerization of fibrin apply combinatory enzyme (TPF) Reptilase from rattlesnake venom of Bothrops atrox. For the diagnosis of lupus coagulants are used fractions poisons Viper Russell Daboja russelli and Taipan Oxyuranus scutellatus containing enzymes that activates factor X and prothrombin, respectively. For determination of Protein C activity is used enzyme "PROTEK" from the venom of the Copperhead snake Ukraine contortrix [Panchenko, H.E., Dobrovolsky A.B. Thrombosis in cardiology. Development mechanisms and therapy. ): Sport and culture, 1999. - 464 C.]. Ancistrini, TPF from the venom of the Copperhead snake Ukraine halys halys, in particular Ancystron-H are used to determine the level of fibrinogen in the plasma of patients receiving hearingcare [Gornitzka O.V., Platonov, T.N., In the Cove GL Enzymes from snake venoms. // Ukr. Bohm. Journe. - 2003. No. 75. Issue 3. - C-32]. In addition, for a more subtle study of hemostasis finds application in a number of other components of the venom of snakes, especially enzymes and disintegrin [Marsh N., Williams V. Practical applications of snake venom toxins in haemostasis // Toxicon. - 2005. - V.45. - P.1171-1181]. The reason for such wide use in coagulatory proteins in snake venoms is their high selectivity towards their targets in the coagulation of blood, while they in contrast to the natural protein effectors are often not inactivated in the natural plasma inhibitors, which is an additional advantage as biochemical tools.

Known combinatory enzyme Ancrod from the venom of the Copperhead snake Calloselasma rodostoma, which is highly specific for the protein of snake venom, which can be used not only as a biochemical tool, but also as a medicine [G.P. Samsa, D.B. Matchar, Williams G.R., Levy D.E. Costeffectiveness of ancrod treatment of acute ischaemic stroke: results from the Stroke Treatment with Ancrod Trial (STAT) // J. Eval. Clin. Pract. - 2002. - V.8. - P.61-70].

Known coagulopathies phospholipase A2 contained in the poisons of snakes [Kini R. Structure-function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms // Toxicon. - 2005. - V.45. - P.1147-1161]. Some of phospholipase A2 can stop the proliferation of tumor cells [Makarova AV, Osipov A.V., Tsetlin, V., Utkin, Y. The effect is of phospholipases A2 from venoms of snakes on neurite outgrowth and survival cell line PC 12 pheochromocytoma of the rat. // Biochemistry. - 2006. No. 71. - S-846], however, none of the known phospholipases is not an inhibitor of thrombin.

Known closest to the stated thrombin inhibitor Petrojarl (Bothrojaracin). It is a preparation of polypeptide nature, isolated from the venom of the rattlesnake, is functionally PETE mixed type (inhibition constant of 15 nm). Structural characteristics (molecular weight, subunit composition, N-terminal amino acid sequence) it belongs to similar to lectins proteins With type [Zingali R.B., Jandrot-Perrus m, Guillin M.-C., Bon .Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition. // Biochemistry. - 1993. - V.32. - P.10794-10802]; differentiating properties.

Mentioned direct thrombin inhibitors do not fully satisfy all the requirements for such substances, there is therefore a need to develop and obtain other thrombin inhibitors (including PETE), has a different mechanism of action and/or a higher selectivity. The relationship between carcinogenesis and hypercoagulability of blood necessitates the search for substances acting simultaneously on both processes.

The invention solves the problem of expanding the range of direct thrombin inhibitors.

The problem is solved due to new connections, highlighted by three-stage liquid chromatography from the venom of E. ipetcco Cobra Naja haje, consisting of a single polypeptide chain with a molecular mass 14350±14 Yes, having seven intramolecular disulfide bonds, N-terminal amino acid sequence NVYQXRKMLQCAMPNGGPF, where X Is Y or W, and possessing antiproliferative action.

First isolated a new substance specifically inhibits thrombin and causes differentiation of tumor cells.

The technical result is to obtain a direct thrombin inhibitor with antiproliferative action.

It is established that the direct thrombin inhibitor isolated from the venom of the Cobra, Naja haje, having N-terminal amino acid sequence NVYQYRKMLQCAMPNGGPF, where X Is Y or W, in addition to the direct inhibitory action on thrombin has specific, i.e. not related to the fact that inhibition of thrombin antiproliferative action. This substance expands the number of biochemical reagents, anticoagulants, and possible drug.

Received direct thrombin inhibitor can be used as a biochemical agent in the study of thrombosis, platelet aggregation, coagulation, proliferation of tumor cells, especially for selective study of the functions of thrombin in hemostasis, or to selectively inhibit the activity of thrombin in the study of other components is the blood clotting system. Diseases that can be treated or examined by using a direct thrombin inhibitor according to the present invention, are: thrombosis, atherosclerosis, restenosis, hypertension, angina, arrhythmia, heart failure, myocardial infarction, glomerulonephritis, thrombosis, peripheral vascular disease, other cardiovascular disease, ischemic, inflammatory disorders, cancer and other neoplastic diseases, and other conditions in which activation of thrombin and its receptor play a pathological role.

Direct thrombin inhibitor according to the invention consists of a single polypeptide chain having a molecular weight 14350±14 Yes according to MALDI mass spectrometry, and seven intramolecular disulfide bonds. This set of structural properties (polypeptide nature, molecular weight within the range of 13-15 kDa N-terminal amino acid sequence of a single polypeptide homologous to the above, 7 intramolecular disulfide bonds) is characteristic only of phospholipase A2 group I (EC 3.1.1.4).

Direct thrombin inhibitor according to the invention is obtained by three-stage liquid chromatography poison Egyptian Cobra Naja haje (figure 1). The first stage of gel filtration media is used with such a pore size that allows the separation fraction with poison compounds 13-15 kDa as from the main toxic fraction of poison (6-8 kDa), and high molecular weight proteins. This carrier may be a resin Sephadex G-50 superfine (Amersham Biosciences) (example 1). The second stage is cation exchange chromatography in a smooth gradient of salt concentration using media carboxymethyllysine active groups, whereby direct thrombin inhibitor according to the invention is separated from the acidic and more basic components of fractions, for example in the column for HPLC Hema Bio CM ("Tessek") (example 2). The third stage - reverse-phase HPLC on a column with the exposed hydrophobic groups, for example with the carrier 18, for the final cleaning of components with close values of isoelectric point and molecular weight, but with a different degree of hydrophobicity (example 3).

Example 1. Conducting gel filtration

800 mg of dried venom of the Cobra Naja haje dissolved in 1.5 ml of deionized water and applied to a column for gel filtration (4,5×150 cm, medium Sepadex G-50 superfine, Amersham Biosciences, Sweden)in 0.1m ammonium acetate buffer, pH 6,2, at a flow rate of 60 ml/hour, the Eluate is collected in tubes fractions of 15 ml, the optical density of the fractions detected at a wavelength of 230 nm.

The collected eluate are combined as shown in figure 1,And concentrate and absoluut by ultrafiltration filter system VivaScience (polysulfone filters Vivaspin20, Sartorius AG, Germany) and liabili irout.

Example 2. Conducting ion-exchange chromatography

Obtained after gel filtration fraction V is applied to the ion-exchange column (8×250 mm, carboxymethylcellulose, DUMB-VIAU, 1000 CM 10µm, Tessek, Czech Republic). The separation is carried out in the buffer 5 mm Tris-HCl, pH 7.5 in the NaCl gradient from 0 to 1M for 100 min at a flow rate of 1.4 ml/min Fractions collected using a collector fractions or manually according to the testimony of the detector at a wavelength of 280 nm. The obtained fractions are concentrated and absoluut filter system VivaScience (polysulfone filters VivaSpin2 and VivaSpin6).

Example 3. Conducting reverse-phase chromatography

Obtained after ion-exchange chromatography of fraction 5 (Fig 1,B) separated on reversed-phase column of 4.6×250 mm, Jupiter 5u, C-18, 300m, Phenomenex, USA) in a gradient of acetonitrile in water from 15 to 45% for 30 min in the presence of 0.1% triperoxonane acid at a flow rate of 1 ml/min Fractions collected using a collector fractions or manually according to the testimony of the detector at a wavelength of 280 nm. From the obtained fractions of the solvents removed by lyophilization. The peak of the target substance is listed below in figure 1,B.

The presence of a direct thrombin inhibitor (PETE) according to the invention in one of the fractions of the venom at each stage of purification is installed functionally after desalting (ultrafiltration) using test thrombin time (the observation is to be pronounced prolongation); other compounds present in the fractions during purification, does not interfere with the determination. The standard test procedure described in example 6.

PETE allocated thus, homogeneous and has a mass of about 14350±14 Yes, that is confirmed by the method of MALDI mass spectrometry with an allowable variation of 0.1% for the range of molecular masses. It has N-terminal amino acid sequence NVYQXRKMLQCAMPNGGPF, where X Is Y or W, established by N-terminal degradation method Edman on automated protein sequencing machine or manually. Polypetala nature of the rest of the PIT is confirmed by means of proteolytic cleavage (example 4). The presence of polypeptide chains 14 cysteine residues, forming 7 intramolecular disulfide bonds, confirmed by total recovery diastereomer reagent with subsequent alkylation (example 5). Basically the nature of the polypeptide compounds is confirmed by the fact of his holding by chromatography on a cation exchange column under conditions described in example 2 (figure 1,B).

Example 4. Holding proteolytic cleavage by trypsin

5 μg of polypeptide compounds with fully restored and alkylated as described in example 5 cysteine residues dissolved in 20 μl of 50 mm phosphate buffer, pH 8.0, add a solution of the trip is in amount to the ratio of enzyme/substrate of 1:50 by weight, incubate 3 hours at 37°C. After desalting mixture is applied to target for removal MALDI spectrum. On the spectrum see the disappearance of the peak with the initial molecular weight and the appearance of several peaks with masses of less than 3 kDa, which indicates the proteolytic cleavage of the rest of the molecule.

Example 5. Total recovery and alkylation of cysteine residues

200 μg of the obtained substance are dissolved in 200 µl of 50 mm phosphate buffer, pH 8.6, containing 6 M guanidine hydrochloride, add 40 ál of 6% solution of a reducing diastereomer reagent (dithioerythritol or dithiotreitol, or mercaptoethanol and the like) and incubated for 12 hours at room temperature. Then add 12 μl of 4-vinylpyridine) - derivatives and incubated for 3 hours at room temperature. Purification of alkylated derivative, which is the main product of the reaction, carried out by reversed-phase chromatography according to example 3. The molecular weight is derived 15820±16 Yes according to MALDI mass spectrometry, which speaks of the unity of the polypeptide chain in the molecule and the inclusion in it of 14 residues pyridylethyl 14 restored cysteine residues. Conduct a similar experience, but without adding a reducing reagent. No change in molecular weight compounds svidetelstvo is about the absence in the original substance of free sulfhydryl groups, i.e. all 14 cysteine residues form in it 7 intramolecular disulfide bonds.

For the selective suppression of the activity of thrombin in vitro experiments can be added to the blood plasma PIT according to the invention to a final concentration of 30-150 nm, while it has no effect on other serine proteases of the blood plasma.

The main functional characteristic of the PIT according to the invention is its ability to reliably above a concentration of 7.5 nm is directly inhibit fibrinogenolytic the activity of thrombin in plasma, which can be verified in the test thrombin time (example 6), in concentrations of up to 47.5 nm it has no significant influence on the readings of the tests prothrombin time and APTT (the difference does not exceed 5% compared with the control experiments) (examples 7 and 8), demonstrating its selectivity against thrombin. When measuring the impact of the PIT according to the invention on amylolyticus the activity of thrombin on chromogenic substrate (example 9) is confirmed by direct inhibition and is set predominantly uncompetitive type of inhibition, which distinguishes it from most other PIT, known from the prior art. Structurally PIT according to the invention is a protein representative of phospholipases A2, has antiproliferative activity against tumor cells (particularly cells FeO is remository rats) and anticoagulant properties.

PIT according to the invention in a concentration of 15 nm prolong thrombin time, plasma human blood 4 times, at a concentration of 30 nm - 8 times, with an increase in prothrombin time and APTT does not exceed 5% compared with the readings of the control experiment in the absence of specified substances (Figure 2). PIT according to the invention inhibits amylolyticus the activity of thrombin in the synthetic chromogenic substrate N-(p-tosyl)-Gly-Pro-Arg-p-nitroanilide (Sigma-Aldrich) with an inhibition constant of about 70 nm. The fact that inhibition of thrombin in the system, prepared without other components of blood plasma, and the absence of amplification inhibition adding small amounts of plasma (0,8% or 3.3%) speaks of the literal nature of the inhibition. Graphics inhibition in the coordinates of Leinweber-Burke in the absence and in the presence of two concentrations of substances converge at one point in the second quarter of coordinates, which indicates a mixed type of inhibition (Figure 3). At concentrations of more than 150 nm (2 µg/l) degree of inhibition of thrombin increases less sharply (see table). At a concentration of 1.5 µm and above PETE reliably stops the proliferation and causes growth narutopedia processes in tumor cells of rat pheochromocytoma line PC 12 (example 10), and in concentrations of up to 15 μm does not cause their death according to the standard MTT-test (table), ie does not render n is them cytotoxic effects, that is a very important factor in the condition of its application in vivo. Cytotoxic properties PETE install on PC 12 cells using the MTT-test according to the method described in the article [Makarova AV, Osipov A.V., Tsetlin, V., Utkin, Y. The effect of phospholipases A2 from venoms of snakes on neurite outgrowth and survival cell line PC 12 pheochromocytoma of the rat. // Biochemistry. - 2006. No. 71. - S-846]: after 24-hour incubation with the test compound, the number of surviving cells was determined by staining 0.05% solution of 3-(4,5-dimethylthiazole-2)-2,5-diphenyl-2H-tetrazole bromide (MTT) for 1.5 hours. The resulting crystals formazan dissolved in dimethyl sulfoxide, measure the optical density on a tablet spectrophotometer (Multiscan) at a wavelength of 540 nm and compared the readings with the readings of the control experiment in the absence of the test compound. In concentrations up to 15 µm PIT according to the invention does not affect the system activation of serum complement by the classical or in a concentration of 1.5 μm for alternative ways that you can define in a standard hemolytic tests based on the lysis of sheep erythrocytes sensitized with antibodies rabbit serum, Guinea pig, or rabbit erythrocytes by human serum, respectively, as described, for example, in [Shoibonov B.B., Osipov A.V., Kryukova E.V., Zinchenko A. A., Lakhtin V.M., Tsetlin V.I., tkin Yu.N. Oxiagin from the cobra Naja oxiana venom is the first reprolysin inhibiting the classical pathway of complement. // Mol. Immunol. - 2005. - V.42. - P.1141-1153], which also suggests that the PIT according to the invention does not affect other serine proteases of the blood plasma. Using the above methods, and techniques described in examples 6-10, establish the selectivity of the PIT, the absence of cytotoxic and the presence of antiproliferative action.

The connection is a direct thrombin inhibitor according to the present invention has anticoagulant activity, and antiproliferative action. It can be used as a biochemical agent in the study of thrombosis, platelet aggregation, coagulation, proliferation of tumor cells. Thus, selective, predominantly uncompetitive nature of direct inhibition will allow more selective study the General stages of the cascade of blood coagulation implemented active thrombin, and to assess the status of the other components of the cascade, such as the contribution of one or another activator or inhibitor of the early stages with the selective inhibition of thrombin by the connection according to the invention. However, changes to the activation of other stages of the cascade connection according to the invention in comparison with the degree of prolongation of thrombin time (and their possible contribution to it) will be minimal (less than 5% at the concentrations specified in PR the measures 6-9).

Example 6. Impact on reading test thrombin time

100 μl of normal citrate plasma is incubated for 5 min at 37°C With 5 µl of 0.0005% aqueous solution of the PIT according to the invention. Then, at room temperature, add 20 μl (1U/ml) solution of human thrombin and note the time of the formation of a fibrin clot on coagulometer or visually. In the control experiment instead of the solution of the compounds according to the invention is injected 5 μl of water. The increase in time the formation of a clot is 800% compared to the control experiment. 100 μl of a 0.3% solution of fibrinogen incubated 5 min at 37°C With 5 µl of 0.0005% aqueous solution of the PIT according to the invention. Then, at room temperature, add 20 μl (1U/ml) solution of human thrombin and note the time of the formation of a fibrin clot on coagulometer or visually. In the control experiment instead of the solution of the compounds according to the invention is injected 5 μl of water. The increase in time the formation of a clot is 800% compared to the control experiment.

Example 7. Impact on reading test APTT

100 μl of normal nitrate plasma is incubated for 15 min at 37°C With 20 µl of 0.0005% aqueous solution of the PIT according to the invention, then add 100 μl of APTT reagent based on ellagic acid and note the time of the formation of a clot. In the control experiment instead of the solution of the compounds according to the invention is injected 20 μl of water. Increase time education the project clot is 0-5% compared to the control experiment.

Example 8. Impact on reading test prothrombin time

100 μl of normal citrate plasma is incubated for 5 min at 37°C With 20 µl of 0.0005% aqueous solution of the PIT according to the invention, then add 100 μl of the reagent on the basis of thromboplastin, and the incubation continued for another 3 min at 37°C. the Reaction coagulation initiated by adding 100 μl of a 20 mm solution of calcium chloride and celebrate the formation of the clot. In the control experiment instead of the solution of the PIT according to the invention is injected 20 μl of water. The increase in time the formation of a clot is 0-5% compared to the control experiment.

The results obtained in examples 7 and 8 demonstrate the selectivity of the PIT according to the invention in respect of thrombin, because it does not affect the activation of the external and internal ways of coagulation and, therefore, on other serine proteases of the coagulation system.

Example 9. Inhibition amylolyticus activity of thrombin

To 100 μl of an aqueous solution of a chromogenic synthetic substrate N-Tos-Gly-Pro-Arg-pNA (Sigma-Aldrich) in several dilutions (from 30 μm to 3 mm) in 10 mm imidazole buffer, pH 7.4, add 20 ál of 0.15 μm and (in parallel the experience) of 0.3 μm solution of the PIT according to the invention, the reaction induce the addition of 20 μl of 0.5 U/ml thrombin. The optical density of the reaction mixture detected at a wavelength of 405 nm every 30 seconds, until it reaches the PLA is O. In the control experiment instead of the PIT according to the invention is injected 20 μl of water. Build three graphs (corresponding to the two concentrations of inhibitor and control) dependence of the reaction rate on the concentration of substrate in the reverse coordinate Leinweber-Burke. The constant and the type of inhibition determined on the basis of the values of angles of inclination and location of the point of intersection of the graphs, respectively.

Because the progress of the tumor and activation of the coagulation system are interrelated, PETE according to the present invention can be used for experiments to study the effect of simultaneous suppression of both processes - proliferation of tumor cells and hypercoagulation.

Example 10. The influence of the PIT according to the invention on the differentiation of tumor cells

Cells of rat pheochromocytoma PC-12 cultured at 37°C and 5% CO2in medium RPMI 1640 (Sigma)containing 15% fetal calf serum (Hyclone) and 2 mm glutamine. To study the action of phospholipases cells scatter in 96-well plate (Corning & Costar) with a density of 5-10·104cells per well (60-70% of the monolayer) and incubated in medium containing 1% serum for 24 hours. Then add the PIT according to the invention in different concentrations and incubation continued under the same conditions. After 24 hours, the morphological changes observed in the growth of neurites, detect visually by microscopy is fixed on the photos. Differentiated cells are, with a length of neurites longer than the length of the cell body. For the quantitative determination neuritogenic activity calculate the percentage of differentiated cells relative to the total number of visible cells (table).

In addition, PIT according to the invention can be used to obtain a pharmaceutical composition comprising a therapeutically effective amount of a direct thrombin inhibitor in a pharmaceutically acceptable carrier, such as lyophilized powder for injection in aseptically sealed ampoule. The drug is readily soluble in water for injection.

Given the effectiveness of the PIT according to the invention in vitro (in a concentration of 30 nm thrombin increases the time for whole plasma 8 times) and the volume of circulating blood in the body, an average single dose can be considered 33-40 mg/kg body weight and the daily dose for the treatment of diseases or conditions listed above, from about 0.01 to 1 mg/kg of body weight per day. Thus, for an average body weight of 70 kg, the dosage level is from about 0.7 to 70 mg drug per day in a single dose or 2-5 fractional doses.

Thus, the invention extends the range of the PIT. Compared with the closest analogue - petroamazonas - declared PIT similar to it and is the source of the selection (snake venom), chemical type (polypeptide connection) and anticoagulant activity (direct thrombin inhibitor mixed type). Direct thrombin inhibitor according to the invention differs from burroarachide structurally that belongs to another class of proteins (phospholipase A2), and functionally that is capable regardless of its thrombin-inhibiting activity to stop the proliferation of tumor cells, inducing their differentiation.

Direct thrombin inhibitor, highlighted by three-stage liquid chromatography from the venom of the Egyptian Cobra Naja haje, consisting of a single polypeptide chain with a molecular weight (14350±14) Yes determined using MALDI mass spectrometry, with seven intramolecular disulfide bonds, N-terminal amino acid sequence NVYQXRKMLQCAMPNGGPF, where X Is Y or W, and possessing antiproliferative action.



 

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15 cl, 1 dwg, 13 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: method of obtaining dodecapeptide of the formula I: H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH and tripeptide of the formula II: X-Asp(Y)-His-Leu-OH is the intermediate compound in its synthesis. Solid-phase synthesis of dodecapeptide I is realised by sequential growth of the peptide chain, beginning with the C-end dipeptidilpolymertill the obtaining of C-end nonapeptidilpolimer, which is condensed with the protected N-end tripeptide of the formula II: X-Asp (Y)-His-Leu-OH where X, Y are protected groups and the obtained dodecapeptidilpolymer is processed with an unblocking agent for removing the protective groups and the polymeric matrix and in 1 stage the end product is given out by means of HELC.

EFFECT: increasing the output of the end product and simplification of the process.

3 cl, 2 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.

EFFECT: improved preparing method, valuable biological and biochemical properties of system.

49 cl, 5 dwg, 2 tbl, 9 ex

FIELD: bioengineering; genetic engineering; medicine; methods of production casamino acids.

SUBSTANCE: the invention is pertaining to the field of bioengineering, genetic engineering, medicine, in particular, to the methods of production of components for nutrient mediums from hydrolysates of animal protein. The invention offers the method of production of casamino acids by the method of the gel permeation chromatography of the hydrolyzed crude acid casein with the contents of the general nitrogen - 0.7-0.95 g in 100 ml of the solution and concentration - 6-10 % on Sefadex G-15, eluating by a distilled water of fractions of an eluate, selection of the active fractions of an eluate by a spectophotometery of portions of the eluate (D254), evaporation of the active fractions under vacuum at the temperature of no more than 55°C. The method allows to simplify the process of production of casamino acids, to reduce its cost and also to obtain casamino acids possessing the high growth- stimulating activity.

EFFECT: the invention ensures simplification of the process of production of casamino acids, reduction of its cost and also production of casamino acids possessing the high growth- stimulating activity.

2 cl, 2 dwg, 1 tbl, 1 ex

FIELD: peptides, pharmacy.

SUBSTANCE: invention relates to a new method for preparing a pharmaceutical composition for the parenteral administration in mammals that comprises salt of difficulty soluble peptides. Method involves treatment of the parent readily soluble acid-additive salt of the basic peptide of LHRH antagonist in the presence of suitable diluting agent with the mixed ion-exchange resin or mixture of acid and basic ion-exchange resins to form free basic peptide. Then ion-exchange resin is removed and free basic peptide is treated with inorganic or organic acid to form the final product, required acid-additive peptide salt followed by addition of suitable pharmaceutical vehicles and/or filling agents and removal of a diluting agent.

EFFECT: improved preparing method.

5 cl, 1 dwg, 1 ex

FIELD: feed mill industry.

SUBSTANCE: method involves treating sunflower oilcake with catholyte; removing treated solution; extracting protein; filtering; drying sediment; grinding; simultaneously with treatment of sunflower oilcake with catholyte, providing treatment of soya with anolyte, with catholyte and anolyte circulating at equal velocities. Apparatus has two chambers connected with each other through semi-permeable partition. Each of said chambers is equipped with electrodes and dc source. Apparatus is further equipped with collecting chamber and pumps with pipelines.

EFFECT: increased efficiency of method and apparatus, reduced production time and decreased costs of additives.

1 dwg, 1 tbl

FIELD: biotechnology.

SUBSTANCE: method involves purification of protein raw, addition of water and ice, milling a mixture followed by hydrolysis. After hydrolysis collagen-containing solution is homogenized and collagen is separated. Hydrolysis is carried out for two stages. The first stage is carried out by treatment of the reaction mixture with lipase from fungus Rhizopus oryzae, and at the second stage a proteolytic enzyme as neutral protease is used. Invention provides preparing collagen approaching to natural collagen by physicochemical and structural-mechanical properties. Invention can be used in food processing industry, cosmetic, medicinal and other branched of industry.

EFFECT: improved preparing method.

4 cl, 3 ex

FIELD: biotechnology, preparative biochemistry.

SUBSTANCE: invention proposes a method for preparing the recombinant human tumor necrosis factor-aplha (TNF-alpha). Method involves culturing the strain-producer E. coli SG20050/pTNF311Δ, disruption of cells by ultrasonic oscillation, extraction of the end protein and 3-step chromatography purification procedure on DEAE-cellulose column in the linear gradient concentrations of NaCl at pH 8.0, on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 8.0 and on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 6.7. The recombinant TNF-alpha prepared by the proposed method shows the reduced content of impurity proteins, nucleic acids and lipopolysaccharides especially that provides its direct medicinal using. Invention can be used in medico-biological industry.

EFFECT: improved preparing method, enhanced quality of polypeptide.

2 tbl, 5 dwg, 2 ex

FIELD: biotechnology, preparative biochemistry.

SUBSTANCE: invention proposes a method for preparing the recombinant human tumor necrosis factor-aplha (TNF-alpha). Method involves culturing the strain-producer E. coli SG20050/pTNF311Δ, disruption of cells by ultrasonic oscillation, extraction of the end protein and 3-step chromatography purification procedure on DEAE-cellulose column in the linear gradient concentrations of NaCl at pH 8.0, on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 8.0 and on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 6.7. The recombinant TNF-alpha prepared by the proposed method shows the reduced content of impurity proteins, nucleic acids and lipopolysaccharides especially that provides its direct medicinal using. Invention can be used in medico-biological industry.

EFFECT: improved preparing method, enhanced quality of polypeptide.

2 tbl, 5 dwg, 2 ex

FIELD: chemistry of polypeptides.

SUBSTANCE: invention relates to a method for space packing (folding) of chemically synthesized polypeptides. Method comprises treatment of polypeptide and/or protein comprising two or more S-butyl-thiocysteine residues with cysteine in buffer providing the folding process at average pH value from 7 to 9 and at temperature in the range from 25°C to 40°C.

EFFECT: improved method for space packing.

19 cl, 13 dwg, 6 ex

FIELD: chemistry of polypeptides.

SUBSTANCE: invention relates to a method for space packing (folding) of chemically synthesized polypeptides. Method comprises treatment of polypeptide and/or protein comprising two or more S-butyl-thiocysteine residues with cysteine in buffer providing the folding process at average pH value from 7 to 9 and at temperature in the range from 25°C to 40°C.

EFFECT: improved method for space packing.

19 cl, 13 dwg, 6 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new N-acylated pseudodipeptides comprising acidic group in neutral or charged form by one end of pseudodipeptide and accessory functionalized branching by the opposite end and corresponding to the general formula (I): wherein each R1 and R2 means acyl group of saturated or unsaturated, linear or branched carboxylic acid comprising from 2 to 24 carbon atoms, unsubstituted or comprising a substitute or substitutes chosen from hydroxyl, alkoxy- and acyloxy-groups; coefficient n has values from 0 to 3; coefficients p and q have values from 1 to 3; coefficient m has values from 1 to 3 except for a case when X means carboxyl or one of its derivative, and in this case it means values form 0 to 3; Y means oxygen atom (O) or -NH; X and Z mean accessory functionalized branching or acidic group in neutral or charged form chosen from the following groups: carboxyl, carboxy-(C1-C5)-alkoxy-, carboxy-(C1-C5)-alkylthio-, phosphono-(C1-C5)-alkoxy-, dihydroxyphosphoryloxy-, hydroxysulfonyloxy-, (carboxy-(C1-C5)-alkyl)aminocarbonyl, (dicarboxy-(C1-C5)-alkyl)aminocarbonyl, (ammonio-(C1-C5)-aminocarbonyl, carboxy-(amino-(C1-C5)-alkyl)aminocarbonyl under condition that at least one substitute among X and Z means an accessory functionalized branching, and their enantiomers and diastereoisomers. Proposed compounds show immunomolulating properties as adjuvants and these compounds can be grafted to antigen to modulate the immune response or can be grafted to pharmacophore to improve the therapeutic effect or its directed delivery.

EFFECT: improved preparing methods, improved and valuable medicinal properties of substances and compositions.

48 cl, 3 tbl, 112 dwg, 5 ex

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