Method for preparing active recombinant proteins

FIELD: molecular biology, medicinal industry.

SUBSTANCE: invention relates to a method for preparing recombinant double-stranded enzyme urokinase (ds-uAP). Method involves culturing cells CHO-Meissi subjected for genetic manipulations that are transfected stably with cDNA encoding pre-prourokinase in culturing medium containing alkanoic acids or their derivatives at temperature from 30°C to 37°C and the following separation the prepared ds-uAP for low-molecular and high-molecular forms. Also, invention involves recombinant ds-uAP, high-molecular ds-uAP and low-molecular ds-uAP in different stages of their preparing. Advantage of proposed method involves the development of method for preparing recombinant forms of mature double-stranded protein ds-uAP and its low-molecular and high-molecular forms.

EFFECT: improved preparing method.

19 cl, 4 tbl, 5 ex

 

The scope of the invention

The technical field of this invention is the preparation of recombinant proteins using genetic engineering of eukaryotic cells.

Background of the invention

Many enzymes or hormones produced by the cell as inactive proenzymes or prohormones and then converted in vivo in place of "use" or "using" in the appropriate active substances. Examples of such enzymes are some metalloprotease matrix, such as collagenase I, plasminogen, chymotrypsinogen, trypsinogen. The specified mechanism of activation is common to hormones such as chromogranin, calcitonin, etc.

Among the molecules that are synthesized in the cell in the form of precursors, a typical example can be considered urokinase (urinary plasminogen activator type-AP). This enzyme is a glycoprotein of plasma, related to the large family of serine proteases. Its main function, along with many other functions, is the physiological activation of plasminogen to plasmin (Barlow, GH, Methods in Enzymology, 45: 239-244, (1976)). The plasmin is a key proteolytic enzyme in the fibrinolytic process, which leads to fibrinolysis (Robbins, K.C. and Summaria, L., Methods in Enzymology, 45: 257-273, (1976)).

The person at-AP is expressed in various tissues in the form of pre-profe is ment (preproorexin) and then secreted into the blood in the form of prourokinase in single-stranded form. So this imagenow form is often abbreviated OC-UAP (single-chain u-PA). OTS-UAP in SDS-page has an apparent molecular mass 50000-54000 daltons and is catalytically inactive.

Blood plasmin provides proteolytic cleavage, which makes proferment OTS-UAP in double-stranded polypeptide, called DC-UAP, which is catalytically and physiologically active.

DC-UAP consists of a - and b-chains, linked together by a disulfide bond. In-circuit carries the catalytic site and also the site of N-glycosylation sites, located at Asn302(A.A. Bergwerff, van Oostrum, J., Kamerling J.P. and J.F. Vliegenthart, Eur. J. Biochemistry, 1995, 228: 1009-1019). Specified form y-AP is called the double-stranded-up with high molecular weight (DC-UAP AMM). Further proteolytic cleavage carried out by plasmin a-chain DC-UAP TIM, leads to the formation of shorter double-stranded molecules of urokinase, called DC-UAP MMOs (double-stranded-up with low molecular weight).

Numerous attempts to obtain DC-UAP through recombinant DNA technologies on the one hand confirmed the suitability of such molecules to the clinic, and on the other hand, the need for this approach, necessary for reasons of safety and cleanliness.

In the patent EP 154272 describes the preparation of recombinant glycosylated OTS-ua is, obtained by introducing the cDNA sequence in the cells of animals.

In the patent EP 303028 describes the preparation of recombinant glycosylated OTS-UAP obtained by introducing genomic sequences in mammalian cells.

Obtaining these patents relates to the recombinant urokinase (OC-UAP) in enzymatically inactive form.

Obtaining an active enzyme (DC-UAP) using recombinant DNA still remains an open question, mainly because of its complex extracellular processing. Actually, in some recombinant eukaryotic systems described to date, a small number of active recombinant DC-UAP. In these eukaryotic systems, the resulting product is a mixture of OC-UAP and DC-UAP (D. Cheng Et al., Chinese Journal of Biotechnology, 1994, 9: 151-159). This mainly is a consequence of the inefficiency of the stages of processing involved in the activation of OC-UAP. This fact raises some issues regarding cleaning two separate forms.

Clinical application of a mixture of UAP-TIM and UAP-MMO would lead to other problems associated with exact reproducibility of the ratio of the respective components, which, in addition, exhibit different pharmacological properties.

On the other hand, obtaining recombinatorial-UAP led to a different strategy of therapy, includes introduction OTS-UAP, as such, then turn the endogenous plasmin active enzyme in the blood stream. In this case, problems are a consequence of the uncertainty of the dose due to the unpredictability of the speed of transformation in vivo.

Currently, however, the molecular form DC-UAP TIM and MMOs are the only molecules that represent a valid interest from the point of view of therapy, based on their successful use for many years for the treatment of disorders of the process of fibrinolysis in the clinic.

Both molecules, which are currently available in the form of pharmaceutical products, are not recombinant (for review see: Scripp's thrombolytic report, PJB Publications Ltd., (1993)). The form of AMM currently obtained by extraction from human urine, as described, for example, in DE 3439980, whereas the shape of MMOs was obtained from cell cultures of embryonic human kidney in the presence of serum, as described in DT 2551017. In this case, the serum provides cells not only by growth factors, but also by plasmin to turn prourokinase in the form of a DC-UAP MMOs. Recently, however, with respect to this product have any worries about security, because of its origin from embryonic cells ("The pink sheet", February 1, 1999, page 6).

The invention

This invention relates to FPIC is Boo obtain Mature recombinant proteins using genetically transfected lines of eukaryotic cells. The process of maturation of the recombinant precursor is achieved by growing the cell line in the presence of alkanovykh acids or their derivatives or their salts for at least 24 hours. According to a preferred variant of the present invention, the Mature recombinant protein is a double-stranded UAP (DC-UAP), expressed from cloned cDNA predecessor.

Therefore, the next subject of the present invention is a method for the isolation and purification of recombinant DC-UAP TIM and MMOs and the products obtained, corresponding to the two forms of Mature urokinase produced according to such methods.

These objects of the present invention provide a method for treating thromboembolic disorders, in which the use of recombinant Mature DC-UAP TIM and MMOs and pharmaceutical compositions containing as active agents recombinant DC-UAP TIM and MMOs, obtained according to the method of the present invention.

Detailed description of the invention

This invention relates to a method for producing a Mature recombinant proteins into the culture environment of genetically modified eukaryotic cell.

Applicants unexpectedly discovered that when genetically modified eukaryotic cells over a period of time at IU is e, equal to 24 hours, treated alkhanovym acids or their derivatives or their salts, which are added to the medium for cell cultures, the transformation of the recombinant protein precursor in the corresponding Mature form is very effective, and the Mature active protein accumulates in large quantities in nadeshiko cell culture.

The conversion of protein-precursor to a Mature form, in particular, is effective in the case where the processing alkhanovym acids or their derivatives or their salts, combined with lowering the temperature of the cell culture to values equal to or below 37°C, preferably constituting from 30°to 36°S, most preferably 34°C, indicating a synergistic effect.

The term "cells subjected to genetic manipulation"refers to cells that were transliterowany or transformed by exogenous DNA, preferably cDNA that encodes a desired protein precursor. In a preferred embodiment, the cDNA sequence is a nucleic acid encoding the precursor of urokinase (preproorexin or PUK). The term "the precursor protein" refers to a polypeptide that is secreted, or otherwise produced by the cell, and which require further proteolytic cleavage or "processing" for ecoactive, or for its products in the form as close as possible to the biologically active and natural form. Other examples of such proteins precursor encoded by the corresponding DNA sequences or cDNA, such Imogene as trypsinogen, chymotrypsinogen, plasminogen, prolidase, prolapse, metalloprotease matrix, i.e. collagenase I, factors related to the cascade of the complement system, and prohormones such as: profactor growth of hepatocytes (Pro-HGF), pre-proinsulin, somatostatin, chromogranin And etc. In a preferred embodiment, the precursor protein is a pre-proferment, and most preferably is pre-PUK or PUK, which becomes active in the form of DC-UAP (double-stranded urokinase). Urokinase (UAP) is a serine proteinase, the main function of which in vivo is the activation of plasminogen to plasmin. In humans, the UAP is expressed in different tissues in the form of pre-proferment, and then secreted into the blood in the form of catalytically inactive prourokinase in single-stranded form (OTS-UAP), which is in turn processed by plasmin in double-stranded UAP (DC-UAP), a catalytically active. DC-UAP built from the a-chain and b-chain, linked together by a disulfide bond, the latter carries the catalytic site. Active DC-UAP the nature detected in blood and urine in two different forms: DC-UAP TIM and DC-UAP MMO which differ in their A-chains, shorter-chain DC-UAP MMOs.

Alcamovia acids or their derivatives or their salts in the prior art up to the present time used in order to increase the output of production/secretion of recombinant proteins in mammalian cell cultures. According to this invention alcamovia acids or their derivatives or their salts act as "amplifiers processing". To the authors ' knowledge the present invention, alcamovia acids or their derivatives or their salts used for the first time as "enhancers processing" protein precursor, as described above.

Alcamovia acid or their salts or their derivatives are preferably a straight chain With2-C10and more preferably C3-C4. Alcamovia acid or their salts and/or derivatives, preferably selected from butyrate or propionate, preferably in the form of their sodium salts, or tributyrin or phenylbutyrate. Especially preferred is sodium butyrate.

Alcamovia acid or derivatives thereof are added to the environment of the cultivation of genetically modified cells in a concentration factor of 0.01 to 500 mm. In the case of mammalian cells are preferred within the concentration range from 0.1 to 20 mm, most preferably 0.5 to 25 mm. However, it will be clear that such concentrations can vary according to the cell line and in accordance with other factors such as the viability of the cell culture during or at the end of processing. According to a preferred variant of eukaryotic cells are mammalian cells selected from the cell, usually used for production of recombinant proteins: cells HEK-293, CV-1, COS, BSC-1, MDCK, A-431, BHK, CHO. In a preferred embodiment, cells SNO represent cells SNO-Messi (ECACC No. 93080520).

As the subject of this invention is a method of obtaining a recombinant DC-UAP human cells SNO, including adding alkanovykh acids or their derivatives or their salts in the culture medium not containing serum, in which the cell line of Cho, genetically modified eukaryotic expressing vector carrying cDNA of preproorexin, is maintained for a time period at least equal to 24 hours at temperatures equal to or below 37°C, preferably in the range from 33 to 35°S, most preferably at 34°C. As the subject of this invention is a method of turning prourokinase (OC-UAP) in the DC-urokinase (DC-UAP) by adding alkanovykh acids or their derivatives or their salts in the environment of the cultivation of cells is BUT the fermentation culture cells at temperatures equal to or below 37°and during the period of time at least equal to 24 hours.

As described above with respect to the natural form for recombinant urokinase, the term "DC-UAP" refers to a catalytically active urokinase, which may be in the form of a DC-UAP TIM and MMOs. DC-UAP TIM and MMOs differ in differential protestirovanny a-chain and show a very similar functional activity. DC-UAP TIM and MMOs can be distinguished from each other, for example, for analysis by different electrophoretic painting in not pampering SDS-PAG: DC-UAP TIM migrates in the field of 50-54 kDa, while the DC-UAP MMO under the same conditions migrates in the field 30-33 kDa.

As the subject of this invention is a method of obtaining a recombinant DC-UAP person who receives the method comprising the following stages:

a) cultivation of genetically manipulated cells SNO, transfected cDNA or genome pre-proc, in culture medium containing alcamovia acids or their derivatives or their salts, preferably butyrate or its salts in a concentration in the range of 0.1 to 20 mm, and at a temperature in the range from 30°to 37°S, even more preferably in the range from 33°to 35°most preferably at 34°;

b) continuation specified under the project over a period of time, at least, equal to 24 hours, or preferably in the range of 48 to 200 hours, most preferably about 120 hours (five days) and

C) obtaining nadeshiko cell culture for isolation of the indicated recombinant DC-UAP person.

In accordance with a preferred variant of the method according to the present invention is a cell line subjected to genetic manipulation, is stable transformant Cho, most preferably the transformant SNO-Messi (ECACC No. 93080520), selected on the basis of acquiring metabolic marker gene. Medium for culturing is preferably culture medium without serum, even more preferably a specific environment, not containing serum and protein, such as commercially available environment. In a preferred embodiment, the medium for culturing cells is CHOMaster®.

Under stable transformants or transfectants SNO mean the clone Cho, transfetsirovannyh expressing vector that stably integrated into the host cell genome. Expressing eukaryotic vector is chosen according to criteria well known in this area: the presence of a strong eukaryotic or viral promoter, such as CMV-IE, late or early SV40 promoter, RSV, to direct the transcription of exogenous DNA; signals the polyadenylation; the transcription enhancers and other regulatory regions, which are selected according to methods well known in the field. Other typical signs expressing vectors are: start replication prokaryotes, gene for selection in eukaryotic and prokaryotic cells, such as gene-lactamase or kanR, or neoR, or tet-F, or hygromycin-R, which is well known to specialists in this field.

According to a preferred variant of the present invention the selection of eukaryotic cells carry out due to the expression of the metabolic marker gene: Trp synthase (gene trpB) Trp-auxotrophic cells SNO, or histidinehydrochloride (hisD gene) on His-auxotrophic cells SNO.

Stable clones preferably chosen on the basis of their growth properties, levels of productivity and stability in culture.

The cultivation of the selected clone SNO usually carried out in a bioreactor according to protocols well known in this field. According to a preferred variant cultivation perform parties. The preferred initial concentration of cells is about 3×105living cells/ml, and viability of cells in the inoculum is usually more than 95%, as measured by the elimination method with dye Trifanova blue. Typically, the ratio of the volume of inocul the same volume of fresh medium" varies from 1:1 to 1:5, and in accordance with the total capacity of the bioreactor and cell growth.

When the density of cells in the range from 1×106up to 4×106cells/ml, usually reached after 3-5 days of growth, cells are separated from the used environment, for example, tangential filtration or centrifugation, and resuspended in the same initial volume of fresh medium to which were added alcamovia acid or their salts or their derivatives in final concentrations ranging from 0.1 to 20 mm. Alcamovia acid or their salts or their derivatives add in the moment of making an inoculum of cells or after that. Adding alkanovykh acids or their salts or derivatives thereof, optionally can be repeated during growth or fermentation. Alcamovia acids, salts and/or derivatives are preferably chosen from: butyrate or propionate, preferably their sodium salts, tributyrin and phenylbutyrate. Especially preferred is butyrate and its sodium salt, which is added in the preferred concentration is from 0.5 mm to 2.5 mm, most preferably from 1 mm to 1.5 mm.

Influence alkanovykh acids on the production of recombinant processioning protein further exacerbating lowering the temperature of the cell culture below 37°C, preferably in the range of temperatures comprising from 30°to 36°With more prefer is Ino 33-35° S, most preferably 34±0,5°C. During fermentation also control glucose levels, and support preferably above 1 g/liter Fermentation batches during the phase of production is preferably carried out in accordance with the following parameters:

Temperature: 34±1°C, preferably 34±0,5°

pH: 7,15±0,1

Rho2: 50±20%

In accordance with the preferred fermentation conditions a higher concentration DC-UAP get through 5 days of cultivation after adding alanovoy acid, derivatives thereof, or their salts. For the production of active product can be monitored using functional or immunological assays. For example, for products DC-UAP can be traced chromogenic analysis, such as analysis of Pefachrome®. In the alternative case, the production of active DC-UAP or disappearance OTS-UAP can be monitored in SDS-page under denaturing and reducing conditions, due to different pattern of migration during electrophoresis: OTS-UAP actually migrates as a single-chain polypeptide ˜50-54 kDa, while the DC-UAP TIM is divided into a-chain and b-chain, respectively, migrating in the region of ˜20 and ˜33 kDa.

According to a preferred variant exhausted adosados cell culture (i.e. the culture medium, which has grown cells)containing the C-UAP, usually harvested after 3-8 days of cultivation, usually on the fifth day, when the balance between the levels of recombinant protein and cell survival (the latter preferably is maintained above 70%) is optimal. Alternative adosados after nutrient depletion collect when OTS-UAP missing as measured in SDS-pampering SDS page, and the maximum is converted into a DC-UAP, where the DC-UAP mean a mixture of DC-UAP TIM and MMOs. Usually the optimum time for the selection of Mature DC-UAP is in the range from 48 to 200 hours, with a preferred period of time is 120 hours of cultivation in the presence of alkanovykh acids or their derivatives or their salts, and usually corresponds to the level of the output DC-UAP about 4000 IU/ml According to the described variants of the present invention the transformation of the forms of the precursor (pre-Pro-UAP, UAP, OTS-UAP) in the catalytically active DC-UAP is characterized by greater than 95% efficiency according to the definition of analytical electrophoresis in pampering SDS-page. Out of the total number of the received DC-UAP about 80% is in AMM-form, and the remaining 20% in MMO form. Therefore, the next subject of this invention is adosados cell culture containing DC-UAP obtained according to the method described and characterized the stages (a) through (C).

In one of the following is the option present invention relates to a method for producing recombinant catalytically active DC-UAP TIM and MMO which is formed in the effective conversion of catalytically inactive OTS-UAP or Pro-UAP or prourokinase directly carried out in a depleted culture medium and characterized by the rate of conversion of the precursor into the Mature protein, which is more than 95%.

The next subject of the present invention is a chromatographic method for the isolation of recombinant forms of the molecules DC-UAP TIM and DC-UAP MMOs, characteristic of which is the use of nadeshiko cell culture obtained in stage C) of the process of obtaining DC-UAP.

According to a preferred variant of the invention DC-UAP MMO is separated from the DC-UAP AMM way, including ion-exchange chromatography, and preferably in accordance with the following additional steps: d) acidification nadeshiko cell culture weak acid to a pH in the range from 5.0 to 5.8, optionally with the addition of non-ionic detergent and filtering, (e) contacting nadeshiko with column for ion exchange chromatography at a pH value in the range from 5.5 to 6.5; (f) the release of DC-UAP MMOs adding a buffer solution with pH value in the range from 5.5 to 6.5, in addition, containing monovalent ion in a concentration of from 200 to 300 mm, as, for example, 250 mm NaCl in phosphate buffer; (g) the release of DC-UAP AMM adding upernova solution at pH values from 5.5 to 6.5, optionally containing monovalent ions at a concentration equal to at least 400 mm, as, for example, 500 mm NaCl in phosphate buffer. During chromatography also hold intermediate rinsing to get rid of all of the components that are not associated with specific DC-UAP TIM and MMOs, and washed with buffers and/or solutions, is well known in this field.

According to another variant, the present invention relates to an additional purification of the recombinant DC-UAP TIM and MMOs until therapeutic level of cleaning, which uses two forms, dedicated, respectively, at stage (g) and (f) of the separation process, and which, moreover, includes affinity chromatography on a column of benzamidine. The specified column allows conducts the cleanup routine to get rid of possible traces of OTS-UAP, if they are present.

Chromatography on benzamidine usually performed according to methods well known in the field. According to a preferred variant of the method of purification of DC-UAP TIM purified by chromatography on benzamidine, optionally subjecting the eluate obtained in stage g) processing the following additional stages: g') contacting the eluate containing DC-UAP TIM with benzamidine column when the pH from 6.2 to 6.8; (g) release of DC-UAP mm buffer solution at znacheniyam in the range of 3.8 to 4.2, also contains: sodium acetate at a concentration of from 50 to 150 mm, NaCl at a concentration of from 300 to 500 mm; g"') do not necessarily further contacting the selected DC-UAP TIM with a column for gel filtration and the release of DC-UAP TIM phosphate or acetate buffer with low salt content, as, for example, 5 mm sodium phosphate buffer, at pH values from 4 to 7.

According to another variant of the present invention DC-UAP MMO purified by chromatography on benzamidine through further processing of the eluate from stage f) in accordance with the following additional stages: f') contacting the eluate containing DC-UAP MMO with benzamidine column at pH values from 6 to 8; (f) the release of DC-UAP MMOs solution at pH values of 3.8 to 4.2, also containing sodium acetate at a concentration ranging from 50 to 150 mm, NaCl at a concentration of from 300 to 500 mm; f"') additionally, optional communication dedicated DC-UAP MMO with a column for gel filtration and the release of DC-UAP MMO buffer solution at pH values of 4 to 7, as for example, 5 mm sodium phosphate or acetate buffer.

The product(you), obtained by combining methods of production (stage a to C), separation (stage d to g) and treatment (stage from g' to g' and f' to f"'), characterized in that used adosados derived from a depleted environment of cell culture, n is obtained in stage C) of the process of obtaining, is a recombinant DC-UAP. Recombinant DC-UAP is a molecular form of mm, as defined previously, and it is obtained when the level of treatment of more than 90%, and/or DC-UAP in MMO form, as defined earlier, when the cleaning level above 90% according to the definition of analytical electrophoresis in SDS-page.

Purified recombinant DC-UAP (AMM and/or MMO) is the active form, which is confirmed by functional and biochemical analyses, and has a therapeutic degree of purification in accordance with the requirements of the European Pharmacopoeia. Therefore requires no further processing and/or purification, in contrast to recombinant Pro-UAP or OC-UAP obtained through recombinant DNA technologies related to the prior art. The molecular form of the product is confirmed by the structural data obtained by a mass spectroscopy and by analysis of the N-end way of degradation on Admino.

As defined by functional analyses, such as analysis of fibrinolysis, determination of the constants of the Michaelis-Menten, analysis of the binding of the inhibitor of plasminogen activator (IAP-1), recombinant products obtained according to this invention, is functionally indistinguishable from the extracted DC-UAP, as their activity in the analysis of fully commensurate with the activities of the former is regionmore natural product.

In addition, recombinant products are mainly derived from cells of Cho, which have well-established reliability in the sense of production of recombinant proteins.

Purified recombinant DC-UAP TIM and MMOs according to this invention is used as a powerful fibrinolytic means for the treatment of thrombosis and pathological phenomena of any other kind, which must pharmacological way to remove the clot plasma. Their application is confirmed by the well-proven clinical application of relevant natural extractable forms.

Therefore, the following variant of the invention relates to recombinant DC-UAP AMM/MMO obtained according to the method of the present invention, for the treatment of thromboembolic phenomena, requiring removal of clots by pharmacological means, such as occlusion of peripheral arteries, clearance of catheter embolism of the lungs, deep vein thrombosis or treatment of myocardial infarction.

The best way to implement the present invention are described by the following experimental examples.

Description of the drawings

Figure 1: revitalizing SDS-page of recombinant DC-UAP.

Shown revitalizing SDS-page of purified DC-UAP, which is obtained after addition of medium with cell culture 1.2 mm of butyrate and fermentation for 5 days.

Under reducing conditions DC-UAP split into A-chain (20 kDa) and b-chain (33 kDa), and OC-UAP migrate to somewhere around 55 kDa. Lane 1: purified urokinase from recombinant culture SNO grown in the absence of Na-butyrate; lane 2: purified urokinase from recombinant culture SNO grown for five days in the presence of 1.2 mm Na-butyrate. The growth conditions described in the text; lane 3: molecular mass standard.

Figure 2: Analysis of the masses of the recombinant and the extracted DC-UAP.

The figure shows the spectrum obtained by mass spectrometric analysis of recombinant and extracted DC-UAP (TIM and MMOs) as in native (glycosylated; two upper spectrum)and deglycosylated forms (two lower spectrum).

Figure 3: Analysis of fibrinolysis.

The experiment was performed by adding 0.5 ml of human plasma to 100 μl of urokinase (1000 U/ml), and incubated at 42°C for 5 minutes To the mixture was added 100 μl of thrombin (20 U/ml), and measured the absorbance at 660 nm for 20 min at 42°C.

Shaded area from left to right in the time point "0 seconds" and the time point "425 sec are: (A) recombinant urokinase; C) extracted urokinase; (C) plasma without addition of thrombin (control without clots); (D) plasma with thrombin, but without the addition of urokinase (positive control image is of clots).

In point 425 sec, there is complete lysis in that case, if the clot is present or recombinant urokinase extracted.

It is shown that recombinant and extracted urokinase (DC-UAP AMM) show the same time of fibrinolysis.

EXPERIMENTAL PART

Example 1: Cloning and selection of stable clones expressing the pre-Pro-CC

The cDNA sequence encoding the pre-PUK person (corresponding to sequence ID D00244 in Genbank), was synthesized from mRNA cell line human kidney (CAKI-1) according to methods well known in the field, are described, for example, in Molecular Cloning: A laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory press, (1989).

Briefly, cDNA was synthesized in the reaction with reverse transcriptase AMV (Bhringer-Mannheim) in the presence of a mixture of mRNA, oligo dT18 (Bhringer-Mannheim) and a mixture of four deoxynucleotides (dATP, dGTP, dCTP, dTTP), according to manufacturer's instructions.

A mixture of cDNA molecules specific amplified in PCR with the following 5'- and 3'-primers:

Oligo (5'):5'TAGCGCCGGTACCTCGCCACCATGAGA3'

Oligo (3'):5'TGGAGATGACTCTAGAGCAAAATGACAACCA3'

Got the cDNA sequence of length 1296 nucleotides encoding the sequence of pre-proc person, and cloned into an integrating expressing vector derived from pBR322 carrying the following items:

- posledovatelno the ü cDNA pre-proc person under the control of the early promoter of SV40 virus (Benoist C. and Chambon P. 1981, Nature 290: 304-310).

- breeding marker TrpB for metabolic selection in cells SNO-Messi (Hartman, SC Mulligan, RC (1988). Proc. Natl. Acad. Sci. USA, 85: 8047-51).

- resistance to ampicillin (bla gene) as a marker of antibiotic resistance in E. coli.

- the beginning of replication of E. coli for amplification in E. coli.

End-obtained recombinant expressing vector called pTZA9.

The cell line Cho used to obtain recombinant DC-UAP, was the cell line Cho-Messi (ECACC, Porton Down, Salisbury, United Kingdom, Reg. No. 93080520).

This cell line is able to grow in suspension in a chemically defined medium CHOMaster® (Ferruccio Messi Cell Culture Technologies, Zürich, Switzerland) without adding any serum or protein component. The doubling time of the specified cell line in such environments (full and selectivity environments) is about 24 hours.

Transfection of cells SNO-Messi vector pTZA9 carried out according to the method described in Felgner et al., (1987). Proc. Natl. Acad. Sci. USA, 84, 7413-7417, and complied with the recommendations of the manufacturer of reagents for transfection. Briefly, 1 µg DNA expressing vector pTZA9 (in a solution of 100 μg/ml) was mixed with 30 μl of Lipofectin® (GIBCO BRL, Life Technologies) approximately 500 μl of exponentially growing cells SNO-Messi (about 1×106cells) Mixture for transfection was kept 30 min at room temperature before adding environment CHOMaster with the addition of 25.1 mg/l tryptophan and incubation over night at 37°C.

The cell line Cho-Messi is auxotrophic for tryptophan, and therefore has an absolute need for the specified amino acid for growth. After transfection cells SNO-Messi vector expressing pre-PUK, also bearing the gene cryptotanshinone, cells SNO-Messi benefit from the acquired ability to produce tryptophan, and now can grow in a chemically defined medium in the absence of tryptophan adding serine and indole.

Stable transfetsirovannyh cells obtained after limiting dilution in selectivity environment CHOMaster® (without tryptophan) with the addition of serine (0.02 g/l) and indole (0.35 g/l).

The selection of clones producing urokinase, was performed by dilution of the mixture for transfection selective environment CHOMaster® the holes for micrometrology. Another system used to obtain the selected individual clones producing urokinase, was a 1:10 dilution of the cell suspension, containing approximately 103cells/ml, a solution of high viscosity, containing 0.2 g/l Methocel in selectivity environment CHOMaster® with the addition of 4% cialisbuynow fetal calf serum. The viscous mixture was sown in the 24-cell and tablets in 2 weeks with a sterile pipette tip was collected from the nutrient clones and began a new culture in suspension.

The following strategy cultivation was adapted for obtaining seed in the bioreactor. Culture of cells grown in the wells for micrometrology, then split 1:3 selective environment CHOMaster® in large cells (24-cell and 6-cell tablet) and then in bottles of 25 and 75 cm2after cell density reached approximately 4-5×105cells/ml

Keeping constant the ratio of dividing into parts (1:3) and the density of the cells in which they are divided into parts in T-flasks, suspension culture was transferred to a rotating vials of 2 l (Integra Bioscience, Switzerland), and during the growth process used working volume of 1 L.

Example 2: determination of the optimal conditions secretion/processing DC-UAP.

First determined the optimal conditions on the concentration of butyrate and temperature upon receipt at the laboratory scale. Preliminary experiments aimed at optimizing the concentration of Na-butyrate, performed in the culture of the recombinant cell line Cho 1 liter (1×106cells/ml)to achieve the best products DC-UAP (TIM and MMOs) and the highest survival rate of cells after 5 days of cultivation in rotating flasks.

Table 1 presents data on the viability of the cells and output from the ADF after 5 days of cultivation in rotating f is akanah 1 liter at 37° With and with different concentrations of Na-butyrate. The activity of the UAP was monitored using the chromogenic analysis, using the chromogenic substrate Pefachrome® UK (54-46) (Pentapharm LTD., Basel, Switzerland)according to the manufacturer's instructions; cell viability was monitored every day and measured with a microscope after exclusion of dead cells by the method of dyeing Trifanova blue, as described in Doyle et al. A, Griffiths, JB and Newell, DG (Eds.) (1994), "Cell and Tissue Culture: Laboratory Procedures". John Wiley & Sons. New York.

Table 1
Na-butyrate in a 1 l culture in a rotating vialsThe activity of DC-UAP (IU/ml)Cell viability
0 (control)32092%
0.6 mm52683%
1.2 mm95976%
2.5 mm36156%

As shown in table 1, the most effective concentration of Na-butyrate in relation to output the UAP after 5 days of cultivation in rotating flasks with a volume of 1 l at 37°1.2 mm. At this concentration, cell viability is still quite high (76%), even in comparison with the control group (92%). High cell viability prevents too much zagryaznennosti fragments of cells and proteins related to the owner, and reduces the load of impurities that must be removed during the cleaning process.

The second series of experiments was performed at a concentration of butyrate, equal to 1.2 mm, with a 5-day fermentation to determine the optimum temperature for the culture of the recombinant cell line Cho volume of 2 l (1,5-2×106cells/ml) in the bioreactor.

Table 2
Temperature (1.2 mm Na-butyrate)The activity of DC-UAP (IU/ml) (5 days of cultivation)Cell viability
37°With (control)183053%
34°504367%
32°80084%

Table 2 shows that 34°is the temperature, which allows to achieve a high level of productivity UAP along with high cell viability. Interestingly, when the same experiment was performed with 32°With activity equal 3961 U/ml was achieved only after 12 days at low cell viability (63%).

Reducing the time of cultivation to a high degree, it is desirable for economic reasons and to preserve the integrity of the protein. In fact, it is desirable to avoid permanent is Noah exposure recombinant product with different proteolytic and glycolytic enzymes, released from lysed cells.

Based on the data shown in table 2, it is clear that lowering the temperature of the cultivation of up to 34°along with the addition of Na-butyrate (1.2 mm) in the cultivation environment, leads to a synergistic effect, resulting in increased output active UAP.

Based on these data we can conclude that the processing of 1.2 mm Na-butyrate at a temperature of 34°C is the optimum condition for obtaining active DC-UAP and viability of cells, in particular during the 5-day cultivation period.

Example 3: preparation of recombinant DC-UAP in the cells of SNO in the bioreactor with a volume of 2 liters

Inoculation of the cells was performed in a bioreactor with a volume of 2.4 l (Infors HT, Type Labforce, Bottmingen, Switzerland) division exponentially growing cell culture. The ratio of the volume of the inoculum volume of fresh medium was selected from 1:1 to 1:5, in accordance with the total capacity of the bioreactor and culture conditions. Chose a working volume of 2 l and the final cell density of the inoculum 3×105cells/ml

The culture used for the primary inoculum had a viability of at least 95%. During growth in suspension culture in the bioreactor was monitored glucose concentration, which never remained below 1 g/l in a depleted environment.

For culture ustanavli the following parameters of fermentation:

Temperature: 37±0,5°

pH: 7,15±0,1

Rho2: 50±20%

When cell density reached values of about 2×106living cells/ml, cells were separated from the depleted environment tangential filtration (or centrifugation). The cells are then resuspendable in the bioreactor in the same initial volume of fresh medium CHOMaster® with the addition of sodium butyrate to achieve a final concentration of 1.2 mm.

The culture temperature was lowered to 34±0,5°and all other parameters of the fermentation was maintained such that set before, as follows:

Temperature: 34±0,5°

pH: 7,15±0,1

Rho2: 50±20%

For the production of active u-AP (KK-UAP TIM and MMOs) was monitored using the chromogenic test with a specific substrate, Pefachrome® UK. Observed a gradual increase in activity up to the maximum values of up to 7000 IU/ml are achieved after 4-5 days of fermentation. At this point, cells were collected and then discarded, and emaciated culture medium containing DC-UAP, then processed for purification.

Average levels of products related to the four independent fermentation presented in table 3:

Activity towards the chromogenic substrate (IU/ml)
Table 3

The results of fermentation
Days after adding 1.2 mm Na-butyrateCell viability
1155±23%87±6%
2662±32%81±12%
31995±13%78±15%
43170±23%72±14%
55043±13%67±10%

In the bioreactor was observed gradual decrease in viability of cells from the first to the fifth day of cultivation after the addition of sodium butyrate. The minimum value of the viability of the cells at 1.2 mm of butyrate was observed on the fifth day of cultivation, and was not significantly below 70%, and therefore, it was still quite high. When these values are assumed to have a limited release degrading lysosomal enzymes into the growth medium.

As shown in table 3, a significant increase in the expression of DC-UAP in the retrieval process, as described, is mostly in the last 2/3 days of fermentation after addition of sodium butyrate. Stability the most part Sekretareva urokinase in emaciated environment optimally limitation 2/3 days at a temperature of 34°C. the Specified combination of parameters reduces the impact on protein degrading AK is Yunosti proteolytic and glycolytic enzymes, and provides high quality recombinant protein purification. Figure 1 shows the products obtained after fermentation in the absence or in the presence of 1.2 mm of butyrate at a temperature of 34°C. Under the latter conditions can be identified complete metamorphosis OC-UAP in DC-UAP compared with culture, to which was added Na-butyrate.

Example 4: Purification of DC-UAP TIM and MMO

Cleaning DC-UAP TIM.

Adosados culture cells grown in the bioreactor, obtained as described in example 3, and acidified by the addition of CH3COOH to pH 5.5, and was purified from residual cells by filtration through a 0.45 µm filter. Added tween-80 to 0.01%, and adosados was applied on the column for ion exchange chromatography (SP Sepharose Big Beads, Amersham-Pharmacia), previously equilibrated to 20 mm buffer solution of sodium phosphate pH 6.0. The column size was 10 cm in height and 2.6 cm in diameter. The flow rate during deposition and leaching was 10 ml/min and during the elution 2 ml/min

After application, the column is first washed with 3 volumes of buffer solution containing 20 mm sodium phosphate, 150 mm NaCl, pH 6.0, in order to remove non-urokinase impurities, and then another 3 volumes of buffer solution containing 20 mm sodium phosphate, 250 mm NaCl, pH 6.0, in order eluted mainly DC-UAP MMOs. Last kept frozen for further purification.

Elution DC-UAP TIM spent passing through the ion exchange column buffer solution containing 20 mm sodium phosphate, 500 mm sodium chloride, pH 6.0.

The resulting eluate containing urokinase (DC-UAP AMM) was brought to pH 6.5 by adding 1 N. NaOH. Then it was applied to the affinity column with benzamidine-separate 6V, pre-balanced at least 2 volumes of buffer solution containing 20 mm sodium phosphate, 400 mm NaCl, pH 6.5.

The column size was 10 cm in height, 2.6 cm in diameter. The flow rate during application, washing and elution was 2.5 ml/min

Then the column was washed with 2 volumes of buffer solution containing 20 mm sodium phosphate, 400 mm NaCl, pH 6.5, and finally have suirable 2.5 volumes of buffer solution containing 100 mm sodium acetate, 400 mm NaCl, pH 4.0.

Column benzamidine allowed to get rid of impurities that are not related to urokinase, as well as from unrecorded amounts, if any, OTS-UAP, which may be present in the downloadable material.

The fractions containing DC-UAP TIM, identified as fractions, related to relatively stable and unique peak on the chromatogram, and United.

Received combined fractions were applied to a column for gel filtration (size-exclusion chromatography-based separation on the size of the molecules). Column to GE the R-filter had a height of 30 cm and a diameter of 2.6 cm, and was previously equilibrated with buffer containing 5 mm sodium phosphate, pH of 4.9. Used a flow rate of approximately 3 ml/min

Thus, DC-UAP urokinase type AMM was suirable in pure form by elution with buffer containing 5 mm sodium phosphate, pH 4,9, as shown in figure 1 (lane 2).

Finally, urokinase was ready for cooking in a buffer appropriate for the final lyophilization.

Cleaning DC-UAP MMOs.

Fraction(AI)corresponding to the DC-UAP MMO collected from washing the ion-exchange column buffer solution containing 20 mm sodium phosphate, 250 mm NaCl, pH 6.0, were combined and then purified passing through affinity chromatographic column with benzamidine. Before applying for the specified column, pooled fractions were brought to pH 6.5 or 7.0 by adding 1 N. NaOH, and the column was pre-balanced at least 2 volumes of buffer solution containing 20 mm sodium phosphate, 400 mm NaCl, pH 6.5. After loading the column was washed with 2 volumes of buffer solution containing 20 mm sodium phosphate, 400 mm NaCl, pH 6.5, and finally, suirable 2.5 volumes of buffer solution containing 100 mm sodium acetate, 400 mm NaCl, pH 4.0. The fractions containing DC-UAP MMOs, identified as fractions, related to relatively stable and unique peak on the chromatogram, and United.

p> Received combined fractions were applied to a column for gel filtration (size-exclusion chromatography-based separation by size molecules) with a height of 30 cm and a diameter of 2.6 cm, pre-equilibrated with buffer containing 5 mm sodium phosphate, pH of 4.9. Used a flow rate of approximately 3 ml/min

Thus, DC-UAP urokinase type MMOs have suirable in pure form by elution with buffer containing 5 mm sodium phosphate, pH of 4.9, and, finally, urokinase was ready for cooking in a buffer appropriate for the final lyophilization.

Example 5: Characterization of recombinant DC-UAP

The processing of the recombinant molecule.

Recombinant DC-UAP TIM characterized in comparative studies with a commercial preparation extracted DC-UAP TIM (Ukidan®, Serono) by mass spectroscopy and functional studies.

Molecular mass.

Data mass spectroscopy, the accuracy of which was varied within +/- 50 Yes up to +/- 100 Yes, have confirmed that the molecular weight corresponded to the molecular mass expected after proper processing of the protein precursor (EC-UAP) and, in particular, as shown in figure 2, confirmed that:

recombinant obtained according to the method of the invention, and extracted commercial DC-UAP VM is very similar in molecular mass, components 48267 and 48565 Yeah, respectively;

in the case of deglycosylation two forms have very similar molecular weight, 46382 (recombinant) and 46313 Yes (extracted).

- similarly, analysis of recombinant obtained according to the method of the present invention, and extracted DC-UAP MMOs were very close to the mass of the glycosylated (33249 and 33189 Yes, respectively) and deglycosylated forms (31029 and 30969, respectively).

In addition, mass spectrometric analysis of MALDI-MS showed that the purified molecules mostly intact (>95%), in other words, it confirms that the degradation products associated with UAP, are present in insignificant quantities, and that, therefore, the selected cleaning methods do not affect the integrity of the recombinant molecule.

The correct processing of the N-end confirmed by way of degradation of Atmano purified DC-UAP TIM. As expected, NH2-terminal sequence of the b-chain is defined as a sequence:

-IIGGEF-,

while NH2-terminal sequence of the a-chain is defined as expected in a sequence:

-SNELHQ-.

The data presented demonstrate that proteolytic cleavage is carried out accurately and in a specific place due Lys158-Ile159and Lys158just removed from the remainder of the molecule. the moreover, analysis peptide mapping confirmed the presence of the correct NH2- and C-ends of both a-and b-chain recombinant DC-UAP TIM.

The nature of glycosylation.

The purified recombinant DC-UAP analyzed glikana using mass spectroscopy and electrophoresis of carbohydrates using fluorescence (FACE). Both methods revealed a stable glycosylation: N-glikana recombinant urokinase consist of fokusirovannyi in the Central part, two-, three - and four-antennal complex chains with a degree of Valerevna 80-90%. Using mass spectroscopy identified the site of glycosylation and confirmed that glycosylation occurs at Asn302.

Functional studies.

Determined the biological activity of recombinant DC-UAP AMM by measuring the following parameters:

- analysis of Kdlinking with the natural receptor for urokinase (see table 4);

analysis the stoichiometric inhibitory activity of an inhibitor of plasminogen activator (IAP-1), which is a natural inhibitor of urokinase;

analysis of the kinetics of inhibition of IAP-1 (see table 4);

- the study of enzymatic parameter Kmwith chromogenic substrates (see table 4);

- the study of the kinetics of plasminogen activation;

- ability to fibrinolysis (figure 3);

- degradation of fibrin.

Table 4SampleK1(M-1·s-1)·10-1Km(mm)Kd(nm)Recombinant DC-UAP TIM2,07±0,570,027±0,00181,24Extracted DC-UAP TIM2,59±0,200,028±0,00501,77

The data shown in table 4 indicate significant functional identity of recombinant DC-UAP AMM obtained according to the method of the present invention, and extracted commercial DC-UAP TIM. In particular, it is shown that the two products are:

(a) similar rate constants (k1education complex with IAP-1, which was measured according Chmielweska et al. Biochem. J. 1988, 251: 327-332,

(b) similar constants of the Michaelis-Menten (Kmfor urokinase substrate, which was measured according to Briggs, GE and Haldane JBS, Biochem. J. 1925, 29: 338-339 and Lijnen, HR et al. Eur. J. Biochem. 1994, 224: 567-574, and

(C) similar constants in the affinity towards the receptor for urokinase (Kd), which was measured according to: Cubellis, M.V. et al., J. Biol. Chem., 1986, 261: 15819-15822.

1. A method of obtaining a recombinant DC-UAP, comprising the following stages:

a) Cultivation of genetically manipulated cells CHO-Messi, stable transfer the aligned cDNA pre-prourokinase, in the medium for the cultivation containing alcamovia acids or their derivatives or their salts at a temperature in the range from 30 to 37°C;

b) the continuation of the specified cell culture during a time period at least equal to 24 h;

c) obtaining the supernatant of cell culture.

2. The method according to claim 1, in which the cultivation of genetically manipulated cells CHO-Messi, stably transfected with the cDNA of preproorexin, in the medium for the cultivation containing alcamovia acids or their derivatives or their salts at a temperature in the range from 33 to 35°C.

3. The method according to claim 1, where the specified period of time at the stage b) is from 72 to 150 hours

4. The method according to claim 1, where the cell viability of the specified cell culture CHO-Messi at the stage b) is at least 70%.

5. The method according to claim 1, where the specified derived alanovoy acid selected from butyric acid, sodium butyrate, sodium propionate, magnesium butyrate, tributyrin, phenylbutyrate in a concentration factor of 0.1 to 20 mm.

6. The method according to claim 5, where at the stage a) specified media for cultivation is a cultural medium that does not contain serum.

7. The method of separation of recombinant DC-UAP TIM and MMOs from depleted environment of cell culture CHO-Messi subjected to gene ingeneri is, which includes stage

d) the acidification of the supernatant of the cell culture obtained according to claim 1, weak acid to pH values in the range from 5.0 to 5.8, optionally with the addition of non-ionic detergent;

(e) passing the acidified supernatant liquid through a column of SP-separate at pH values ranging from 5.5-6.5;

f) elution DC-UAP MMOs adding a buffer solution with pH value in the range from 5.5 to 6.5, containing monovalent ion in a concentration of from 200 to 300 mm;

g) elution DC-UAP AMM adding a buffer solution with pH value of 6-7,5 containing monovalent ions at a concentration at least equal to 400 mm.

8. The method according to claim 7, where the acidified supernatant at a stage d) optionally filtered.

9. The method according to claim 7, where the eluate from step (f) or (g)in addition, purified using chromatography on benzamidine.

10. The method according to claim 9, where for purification of recombinant DC-UAP AMM method includes an additional stage

g') contacting the buffer solution from step g)containing suirvey DC-UAP AMM, with benzamidine column at pH values in the range from 6.2 to 6.8;

g) elution DC-UAP mm buffer solution at a pH value in the range of 3.8 to 4.2, in addition, containing monovalent ions in a concentration of from 300 to 500 mm;

g"') neo is Astelin further contacting lirovannomu DC-UAP TIM with a column for gel filtration, and elution DC-UAP mm buffer solution with a low content of salts at pH values ranging from 4 to 7.

11. The method according to claim 9, where for purification of recombinant DC-UAP MMOs the way, also includes an additional stage

(f) contacting the solution containing suirvey DC-UAP MMOs, obtained in stage f), with benzamidine column at pH values ranging from 6 to 8;

f) elution DC-UAP MMO buffer solution with pH of 3.8 to 4.2, in addition, containing monovalent ions in a concentration in the range from 300 to 500 mm;

f) in addition, optional communication lirovannomu DC-UAP MMO with a column for gel filtration and elution DC-UAP MMO buffer solution with a low content of salts at pH values ranging from 4 to 7.

12. Recombinant DC-UAP obtained by the method according to claim 1.

13. Recombinant DC-UAP obtained by the method according to claim 5.

14. The recombinant product DC-UAP TIM, obtained by the method according to claim 7.

15. The recombinant product DC-UAP MMOs, obtained by the method according to claim 7.

16. The recombinant product DC-UAP TIM, obtained by the method according to claim 9.

17. The recombinant product DC-UAP MMOs, obtained by the method according to claim 9.

18. Recombinant purified DC-UAP TIM, obtained by the method according to claim 10.

19. Recombinant purified DC-UAP MMOs, obtained by the method according to claim 11.



 

Same patents:

FIELD: genetic and protein engineering, medicine, molecular biology, pharmacy.

SUBSTANCE: invention proposes a modified form of plasminogen urokinase type activator (activator) wherein amino acid sequence differs from that in the natural activator as result of replacing the sequence Arg-Arg-His-Arg-Gly-Gly-Ser in the composition of inhibitory loop for the sequence Arg-His-His-Ala-Gly-Gly-Ser and by replacing 24 N-terminal amino acids for the foreign sequence consisting of 16 amino acid residues. Invention proposes the constructed recombinant plasmid (pUABC 34) comprising DNA fragment that encodes a new activator. As result of transformation of E. coli K-12 JM109 cells with plasmid pUABC 34 the recombinant strain E. coli VKPM-8145 as a producer of a modified form of activator is obtained. This polypeptide is characterized by reduced sensitivity to effect of inhibitor PAI-1 and absence of some by-side effects in the complete retention of biological activity of the natural activator produced by the recombinant. This provides effective applying a new activator as a component of pharmaceutical compositions eliciting thrombolytic effect.

EFFECT: valuable medicinal properties of activator and pharmaceutical composition.

6 cl, 6 dwg, 8 ex

The invention relates to pharmaceutical industry and relates to a method of obtaining Mature recombinant protein DC-UAP

The invention relates to bifunctional options urokinase with improved fibrinolytic properties and inhibiting thrombin action, used in obtaining these polypeptides plasmids, as well as thrombolytic means that as biologically active substances contain bifunctional urokinase variant

The invention relates to genetic engineering, in particular to a technology for obtaining high-yielding strains Eserichia coli - produced recombinant human proteins used in modern medicine as thrombolytic agents

The invention relates to biotechnology and allows you to get the polypeptides used as plasminogen activators having high specific amylolyticus and fibrinolytic activity

The invention relates to the production of tissue plasminogen activator (enhanced APT) with prolonged biological half-life existence, increased resistance to heat and acids and effective as an inhibitor of inflammation around the site where the formed thrombus

The invention relates to Enzymology, particularly to a method of increasing the stability of the enzyme urokinase to heat, and can be used for inactivation of viruses present in the drug

FIELD: genetic and protein engineering, medicine, molecular biology, pharmacy.

SUBSTANCE: invention proposes a modified form of plasminogen urokinase type activator (activator) wherein amino acid sequence differs from that in the natural activator as result of replacing the sequence Arg-Arg-His-Arg-Gly-Gly-Ser in the composition of inhibitory loop for the sequence Arg-His-His-Ala-Gly-Gly-Ser and by replacing 24 N-terminal amino acids for the foreign sequence consisting of 16 amino acid residues. Invention proposes the constructed recombinant plasmid (pUABC 34) comprising DNA fragment that encodes a new activator. As result of transformation of E. coli K-12 JM109 cells with plasmid pUABC 34 the recombinant strain E. coli VKPM-8145 as a producer of a modified form of activator is obtained. This polypeptide is characterized by reduced sensitivity to effect of inhibitor PAI-1 and absence of some by-side effects in the complete retention of biological activity of the natural activator produced by the recombinant. This provides effective applying a new activator as a component of pharmaceutical compositions eliciting thrombolytic effect.

EFFECT: valuable medicinal properties of activator and pharmaceutical composition.

6 cl, 6 dwg, 8 ex

The invention relates to genetic engineering, in particular to a technology for obtaining high-yielding strains Eserichia coli - produced recombinant human proteins used in modern medicine as thrombolytic agents

The invention relates to biotechnology and allows you to get the polypeptides used as plasminogen activators having high specific amylolyticus and fibrinolytic activity

The invention relates to the production of tissue plasminogen activator (enhanced APT) with prolonged biological half-life existence, increased resistance to heat and acids and effective as an inhibitor of inflammation around the site where the formed thrombus

The invention relates to genetic engineering, in particular the production of plasminogen activator in cells E

FIELD: medicine.

SUBSTANCE: invention proposes an agent for correction of disturbances in production of cytokines and markers of activation of immune system cells in chronic iron-deficient anemia. Agent represents Sorbifer Durules known early as preparation used in treatment of iron-deficient anemia. Invention provides recovering the level of anti-inflammatory cytokines (IL-1, IL-6, IL-8, FNO-α, INF-α and INF-γ) and normalization of cellular immunity by indices of markers CD-25, CD-71 and CD-34.

EFFECT: valuable medicinal properties of agent.

2 tbl, 1 ex

Hemostatic glue // 2257901

FIELD: bleeding suppression facilities.

SUBSTANCE: invention provides hemostatic glue capable of stopping bleeding and joining tissues, which is aqueous solution containing sodium alginate, acetic acid, and cellulose in specified proportions. Formation of tight film on wound surface takes 62 sec.

EFFECT: enabled application of glue onto wound from one syringe, while uniformly filling wound surface without drying of the latter.

3 ex

FIELD: veterinary.

SUBSTANCE: agent is based on complex compound of colloidal iron liganded by polyoxymethylene-lauric alcohol and contains following components, mg: colloidal iron 50.0-60.0, lauric alcohol 0.1-0.3, copper 0.1-0.3, zinc 0.1-0.3, cobalt 0.1-0.3, selenium 0.001-0.005, and water to 1000. Agent can further contain 30.0-50.0 mg glucose and 0.1-0.6 mg polyvinyl alcohol.

EFFECT: enabled therapeutical and preventive efficiency at low toxicity, the same dispersity, and high stability upon storage.

3 tbl, 7 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention describes benzamidine derivatives of the general formula (I): wherein R1 means hydrogen atom, halogen atom, (C1-C6)-alkyl or hydroxyl; R2 means hydrogen atom or halogen atom; R3 means (C1-C6)-alkyl possibly substituted with hydroxy-group, alkoxycarbonyl-(C3-C13)-alkylsulfonyl, carboxy-(C2-C7)-alkylsulfonyl; each among R4 and R5 means hydrogen atom, halogen atom, (C1-C6)-alkyl possibly substituted with halogen atom, (C1-C6)-alkoxy-group, carboxy-group, (C2-C7)-alkoxycarbonyl, carbamoyl, mono-(C2-C7)-alkylcarbamoyl, di-(C3-C13)-alkylcarbamoyl; R6 means heterocycle or similar group; each among R7 and R8 means hydrogen atom, (C1-C6)-alkyl or similar group; n = 0, 1 or 2, or their pharmacologically acceptable salts, esters or amides. Compounds elicit the excellent inhibitory activity with respect to activated factor X in blood coagulation and useful for prophylaxis or treatment of diseases associated with blood coagulation.

EFFECT: improved method for prophylaxis and treatment, valuable medicinal properties of compound.

26 cl, 2 tbl, 253 ex

FIELD: medicine, clinical oncology.

SUBSTANCE: one should perform blood exfusion at the quantity of 500-550 ml. Removed blood volume should be compensated with crystalloids at the ratio of 1:1.2 - 1: 1.3. Erythrocytic mass should be supplemented with 5-10 ml "Essentiale" solution. This mass should be introduced for a patient at simultaneous ultraviolet blood irradiation for 20-30 min. Plasmapheresis in combination with ultraviolet blood irradiation should be carried out every other day, about 2-4 seances/course, totally. The method provides normalized level of blood leukocytes and body detoxication, it, also, excludes prophylactic course of antibioticotherapy that enables to continue terms of therapy and not to decrease the dosages of chemoradiation therapy.

EFFECT: higher efficiency of therapy.

1 ex, 2 tbl

Hemostatic glue // 2256448

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to hemostatic glue able to stop bleeding and to glue tissues. Glue represents an aqueous solution containing 1-10% of sodium alginate, 0.1-3% of acetic acid and 0.1-15% of feracryl. Applying glue provides ceasing bleeding by formation film after contact with blood and tissues for 30 s. Glue can be stored at room temperature for 3 years.

EFFECT: valuable medicinal properties of glue.

3 ex

FIELD: medicine, diagnostics.

SUBSTANCE: it is necessary to detect pulsing points beyond umbilical ring to perform manual impact upon them, additionally, one should apply medicinal leeches onto areas of umbilical ring, pulsing points, anus and for projection of a sick organ. Moreover, the areas mentioned should be affected once, not less during a course that enhances blood and lymph outflow in disease focus.

EFFECT: higher efficiency of therapy.

1 ex

Up!