The dna fragment encoding the polypeptide kr properties of plasminogen activator, polypeptide kr properties of plasminogen activator, recombinant plasmid dna ra.3 encoding a polypeptide kr properties of plasminogen activator and a pharmaceutical composition having fibrinolytic activity

 

(57) Abstract:

Usage: biotechnology, genetic engineering. The inventive obtained polypeptide KR properties of plasminogen activator by transforming bacterial strain recombinant plasmid DNA RA.3, comprising a DNA fragment encoding the specified polypeptide and having the following properties: fibrinolytic activity of 550,000 200000 IU/mg; molecular weight 38500 2000 Yes; increased half-life compared to the natural counterpart; fiberisima stimulation; thrombolytic activity; solubility in the solution of arginine at a concentration of 10 to 1000 mmol/l when the purity of the product is 95%; the resulting pharmacological composition containing a specified polypeptide in an effective amount and a pharmaceutically acceptable carrier. 4 S. p. f-crystals, 7 ill., 10 table.

The invention relates to a new derived tissue plasminogen activator (t-PA), a DNA sequence that encodes a new-t-PA derivative, expression plasmids containing coding for t-PA-derived DNA sequence, the method of obtaining a t-PA derivative and composition with fibrinolytic activity, containing the t-PA derivative.

Vernuvshayasya fibrin is dissolved due to the sequence of reactions with participation of the fibrinolytic system. The Central reaction is the activation of plasminogen to plasmin, which is caused, for example, tissue plasminogen activator (t-PA). The plasmin, in turn, dissolves fibrin.

The enzymatic activity of natural or acquired genetic method in eukaryotic cells t-PA, namely the catalytic activation of plasminogen to plasmin in the absence of fibrin or cleavage products of fibrinogen is very minor, however, in the presence of these products may increase by about an order.

Blood t-PA under the action of proteases split into A - and B-chain. Both formed the chain remain linked through cysteine bridge. The ability to stimulate the activity of t-PA is a decisive advantage compared with other known activators plasminogen, such as urokinase or streptokinase (M. Hoylaerts and etc., J."Biol. Chem". 257(1982), 2912 - 2919; W. Nieuwenhuizen and others, Biochem Biophys.Acta, 755, (1983) 531 - 533).

The mechanism of action of t-PA in vivo are described, for example, in Korniger u Collen, Thromb. Hamostasis, 46 (1981), 561-565. Inherent in t-PA activity allows you to use it as a tool to combat pathological conditions associated with formation of blood clots (EP-A 0302456, CL C 12 N 9/54, 1989, prototype), Thu ሺ/P> However, the disadvantage of t-PA should be considered a rapid decrease of its concentration in plasma clearance. The consequence of this is that you need a relatively large amount of t-PA to obtain in vivo effective lizirovania of blood clots. High therapeutic doses have often side effects, in particular causing bleeding.

In the European patent N 019620 described the natural breakdown product of t-PA, which contains only domains protease, cringely-11 and N-the end begins with alanine 160 (specified in Pennica, etc. in Nature 301 (1983), 214 - 221, amino acid sequence).

The rate of clearance of this degradation product of t-PA, however, is not significantly different from that of natural t-PA. Only through chemical modification of the catalytic region due to the introduction of blocking groups this indicator can be improved.

The objective of the invention is the change in t-PA so that the resulting derivative had a greatly reduced rate of clearance (longer half-life in blood plasma), should remain lyse clots action, and stimuliruemoe fibrin.

The subject of the invention is therefore modified tissue actiology amino acids (see table. 1).

which at the amino-end may also have additional Met.

As it turned out, the deletion of a portion existing in the native t-PA fields has no effect on the thrombolytic efficacy of protein and activity-dependent fibrin stimulation mutein, which is comparable with that of native t-PA. However, it was found that the proposed according to the invention the t-PA derivative lacking the ability to bind fibrin, but despite this, fibrinolytic activity in vivo is not only not reduced, but even significantly improved as compared to that of native t-PA. Unexpected is the fact that with the introduction of tromboliticaskie effective dose derived there are almost no impact on system fibromas.

Thus, it appears that proposed according to the invention the t-PA derivative has a typical native t-PA specificity for fibrin under physiological conditions. These results were obtained in pharmacological studies proposed according to the invention the t-PA derivative (examples 6 and 7). In addition, the proposed protein has a very high specific (specific) activity. When using the following method denaturirovannyj receiving the th which encodes proposed according to the invention t-PA-proizvodstve and has the nucleotide sequence (see tab. 2).

Proposed in the invention the DNA sequence used in the composition of the recombinant vector for the expression of the new t-PA derivative. Recombinant plasmid for t-PA expression is the next subject of invention. This plasmid expression may also include a DNA sequence that caliroa proposed in the invention of the t-PA derivative, may deviate from the specified DNA sequence within the degeneracy of the code.

The expression plasmid contains, along with the encoding t-PA-derived sequence of a regulated promoter (e.g., tac), effective terminator (e.g., td), a selective marker (e.g., gene-lactamase) and the starting area replication.

A specific example of such a plasmid is pA 27.3, receipt of which is described in example 1.

It is clear that the selection of the plasmid in which to embed the encoding proposed in the invention of t-PA-derived DNA sequence, depends on used later for the expression host cells. Suitable plasmids, as well as minimum requirements that the present is ecialist. Instead of plasmids can also be used for kosmidou, replicative denitive forms of phages ( , M13) and other well-known specialist vectors. Methods of site-directed mutagenesis (site-specific mutagenesis described Morinaga, etc., Biotechnology 21 (1984), 634.

It is preferable to obtain proposed in the invention of the t-PA derivative as the host cells used prokaryotic cells. This formed the so-called "intracellular Taurus" (insoluble protein aggregates) are first separated from the soluble particles, cells, solubilizing under reducing conditions by treatment with guanidine-hydrochloride, then treated with GSSG and, finally, centurybut t-PA is derived by adding L-arginine and GSH. The exact methods of activation of t-PA from intracellular Taurus described, for example, in European patent a-O 219 874 and About 241 022. However, you can use any other way to obtain active protein from intracellular Taurus".

In the case proposed in the invention of the method for cleaning the derived t-PA K2P preferably operate in the presence of L-arginine, usually at a concentration of 10 to 1000 Mmol/l

Proposed in the invention, the separation from the foreign protein by bireysel column (ET1 = Eritrean-trypsin-inhibitor). While ET1 is fixed on a material carrier, for example, on sepharose. Purification via ET1-adsorbing column has the advantage that the material ET1-adsorption column can be loaded directly from the concentrated mixture denaturirovannyj even in the presence of high concentrations of arginine how to 0.8 mol/L. Because of this avoid aggregation K2P, which occurs at low concentrations of arginine (below 10 mmol/l). It is advisable to decontaminate K2P through ET1-adsorbing column in the presence of 0.6 - 0.8 mol/l arginine. Preferably, containing K2P solution had a pH above 7, and particularly preferably of 7.5 and 8.6.

Elution of the product with ET1-column is carried out by lowering the pH, both in the presence and in the absence of arginine, in conditions which ensure good solubility of K2P. The preferred pH in the elution lies in the acidic region, particularly preferably in the region of 3 to 5.5.

Thus obtained K2P has specific t-PA activity 550.000200.000 IU/mg with a purity of more than 95%, preferably above 99%.

According to the invention is obtained t-PA derivative, which has a distinctly longer retention in the plasma due suitable for use as a drug. For example, the need for thrombolytic therapy using K2P dose is reduced at least by a quarter compared with a dose of native t-PA. When using the same doses K2P and native t-PA in the first case, the clotting system is less affected, and bleeding time in contrast to the observed with native t-PA, is lengthened slightly. Thus, the probability of complications due to bleeding during therapy with K2P decreases.

These properties offered by t-PA-derived make it especially suitable for use in farbkomposition, which constitutes another subject of the invention. In the application proposed in the invention of t-PA is derived in the form of a drug to achieve the same with native t-PA efficiency requires a much lower dose injection.

In the drawings showing the following: Fig. 1 is a schematic depiction of obtaining plasmids pA27.3; Fig. 2 - comparison of the binding of fibrin proposed in the invention of the t-PA derivative (curve 1) and obtained in CHO-cells of the native t-PA derivative (donativum t-PA, the split in the physiological cleavage site Arg 275-1le276 [curve 2) and single-stranded t-PA (curve 3)];

Fig. 3 and 4 are diagrams yseR); curve 1: K2P dose of 200 000 IU/kg = 0.25 mg/kg; intravenous infusion over 30 min; the number of test animals (rabbits) = 4; curve 2: ActilyseRdose of 200 000 IU/kg; intravenous infusion over 30 min; the number of test animals (rabbits) = 6;

Fig. 5 - comparative curves actions doses (for rabbits) thrombosis t-PA derivatives (shown as average +SEM: 1kU = 1000 1uU; curve 1: K2P; curve 2: ActilyseR);

Fig. 6 - temporal dependence Simplate bleeding time (BT) before and after a single intravenous injection of placebo or increasing doses of ActilyseRunder anesthesia dogs;

Fig. 7 - the time dependence Simplate bleeding time (BT) before and after a single intravenous injection of placebo or increasing doses of K2P under General anesthesia in dogs.

Example 1. Construction of plasmids pA27.3. The original plasmid pREM7685 described in European patent a-O 242 836, contains the following elements: tac-promoter, the lac region-operator with ATG-start codon, the site encoding the t-PA derivative FK2P, the terminator of transcription from pKK223-3, a gene Actilyse-lactamase gene registertest to kanamycin and site start replication from the plasmid rsus. The sequence of t-PA-derived FK2P formed from nucleotides sequence, described in Nature, 301 (1983) 214 - 221.

For deletion of the F domain of FK2P constructs plasmids pREM7685 used method of Morinaga al. Biotechnology, 21 (1984) 634. For the formation of heteroduplex of pREM7685 isolate two fragments. Fragment A: pREM7685 digested with restriction enzymes EcoR1, the fission products separated by gel electrophoresis and more EcoR1 fragment elute from the gel. Fragment B: plasmid pREM7685 linearized with restriction enzymes Xho I linearized plasmid also get preparative gel electrophoresis. For mutagenesis synthesize the following oligonucleotide:

5 TG TCT TAC CAA GGA AAC AGT GA 3'

For the formation of heteroduplex fragment A, fragment B (450 f mol) and oligonucleotide (75 p mol) are mixed and in the presence of 50 mmol/l NaCl, 10 mmol/l Tris/HCl, pH 7.5 and 10 mmol/l MgSO4incubated for 3 min at 100oC and transferred to ice. Resaturate DNA occurs for 30 min at 60oC. For reparative synthesis to heteroduplex add deoxynucleotides (0.25 mmol/l, ATP (1 mmol/l), NaCl (100 mmol/l), Tris-HCl, pH 7.5 (6.5 mmol/l), MgCl2(8 mmol/l) Actilyse-mercaptoethanol (1 mmol/l), fragment maple DNA polymerase from E. coli (a 0.125 u/µl mixture) and T4 ligase (0.1 units/μl of the mixture). Reparative SinTe the ministries of foreign Affairs and transformants by adding to the nutrient medium 25 μg/ml kanamycin.

Using the method of hybridization of colonies when using the above oligonucleotide as a probe can be selected clones carrying the plasmid pA27.3, which encodes proposed in the invention of t-PA-derived CR. This plasmid differs from the original plasmid pREM7685, lack of customers > PST and SspI site. These both section are contained in the scope of the original plasmid, which encodes the F-domain. Construction of plasmids pA27.3 schematically presented in Fig. 1.

Example 2. Getting active t-PA-derived CR from E. coli:

(a) lysis of the cells and obtaining intracellular Taurus IB S

1.6 kg of wet cell mass of E. coli DSM 3689, transformed with plasmids pA27.3 suspended in 10 l of 0.1 mol/l Tris-HCl, 20 mmol/l EDTA, pH 6.5, at 4oC. To this mixture is added 2.5 g of lysozyme and incubated for 30 min at 4oC; the cells are then transferred to suitable for processing of the form by dispersion at high pressure. To the resulting solution was stirred into 5 liters of 0.1 mmol/l Tris-HCl, 20 mmol/l EDTA, 6% Triton X100, 1.5 mmol/l NaCl, pH= 6,5, and incubated for 30 min at 4oC. Then carry out the separation of the insoluble components (IB s) by centrifugation using a centrifuge Padberg.

The residue after centrifugirovania by subsequent centrifugation.

b) Solubilization 1B

100 g of 1B (wet weight) are suspended in 450 ml of 0.1 mmol/l Tris-HCl, 6 mol/l guanidine-hydrochloride, 0.2 mmol/l DTE, 1 mmol/l EDTA, pH 8.6 and stirred at 25oC for 2.5 h

After establishing pH 3 using HCl (25%) carry out dialysis against 10 mmol/l HCl (3 50L, 24 h, 4oC).

C) Derivatization

Enter the guanidine-hydrochloride (solid) so that the final dilution of the above dialysis 10 mmol/l HCl, the concentration of guanidine-hydrochloride was 6 mmol/L.

The reaction mixture was incubated at 25oC for 1.5 h, then enter the oxidized glutathione GSSG to 0.1 mol/l Tris-HCl (0.05 mmol/l, and pH was adjusted to titer up to a pH of 9.3 by using 5 mmol/l NaOH. The mixture is stirred for 3.5 h at 25oC.

After establishing pH 3 using HCl (25%) carry out dialysis against 10 mmol/l HCl (3 (100 l) and 48 h, 4oC). After dialysis, the reaction mixture was centrifuged and the clear supernatant liquid is treated next.

d) Resaturate

The reaction vessel with a capacity of 10 l fill with a mixture of 0.1 mmol/l Tris-HCl, 0:8 mmol/l L-arginine, 2 mmol/l GSH (reduced form), 1 mmol/l EDTA, pH 8.5. Depending on the conditions denaturirovannaya carried out at 20oC by Tigrovaya get material with a specific activity of 1500 - 10000 1U (mg) definition (example 46). Unit 1U - unit activity, by definition, the WHO, the National Institute for biological standards and control.

d) Concentration denaturirovannah reaction mixture

Denaturirovannogo mixture, if necessary, can be concentrated through hemodialysate.

Example 3. Cleaning KR from E. coli.

1. Cleaning KR from E. cjli by affinity chromatography on ET1 - sepharose after pre-concentration

(a) Elution using citric acid

The mixture after denaturirovannyj concentrate through a Memo, dialysator (Asahi AM 300) 1: 23 and supplemented with 0.5 mmol/l Balanced mixture of 0.1 mmol/l Tris-HCl, pH 7.5, 0.8 mmol/l arginine, 0.5 mmol/l NaCl column with ET1 (Eritrean-trypsin-inhibitor)-separate (volume = 50 ml) load 550 ml of the concentrate is subjected to repeated oxidation mixture (10 column objects/h to 10 SV/h) and washed with equilibrating buffer until until the absorbance at 280 nm reaches the value buffer. Elution of the bound material is carried out by using 20 mmol/l citric acid, pH of 3.2 (see tab. 3).

b) Elution with 0.3 mmol/l arginine, pH 4.5

Denaturirovannogo the reaction mixture is concentrated, as described with ET1-separate (25 ml) load 800 ml concentrate (12 BV/h) and washed with equilibrating buffer until until the extinction of the eluate at 280 nm does not reach the extinction of the buffer. Associated material elute with 0.3 mmol/l arginine, pH 4.5, (see tab. 4).

2. Cleaning KR from E. coli by affinity chromatography on ET1-sepharose without prior concentration.

Balanced using a mixture of 0.1 mmol/l Tris-HCl, pH 7.5, 0.8 mmol/l arginine, 0.5 mmol/l NaCl column ET1-separate (volume = 10 ml) load 12 l re-oxidized mixture and washed with equilibrating buffer until such time as the extinction of the eluate at 280 nm reaches the value of the extinction of the buffer. Elution of the bound material is carried out using 0.8 mmol/l arginine, pH 5 (see table. 5).

Example 4. Expression KR gene using other vectors. The sequence colorous CR, clone in cosmides pHC79. Kosmidou, as described by Collins, J., and others in the PNAS USA 75 (1978) 424, Packed in Actilysephage transducer in E. coli ED 8654 and Express.

The protein purified as described in example 3.

Similarly clone K2P gene in M13 vectors+, MP18, M13-, MP19, pUC18, pUC19, pBR322, pBR328 and pSP64.

Molecular weight, amino acid sequence and the activity was determined as described below.

Use ografia high-pressure reversed-phase.

The homogeneity of purified using affinity chromatography with ETI-separate material shown using SDS - PAGE and liquid chromatography high-pressure reversed-phase (R P-HPLC). The relative length of the run for K2P from prokaryotes calculated molecular weight 38,550 + 2,000 Yes. Densitometric evaluation gives purity of the drug is above 95%.

RP-HPLC are based on different interaction of proteins with hydrophobic matrix. This family is used as an analytical method for the qualification degree of purity.

Analysis of the purified K2P from E. cjli passes through the separation column Nucleosil 300 (Kduer) using a gradient triperoxonane acid - acetonitrile (buffer A: 1.2 ml triperoxonane acid in 1000 ml of water; buffer B: 300 ml of water, 700 ml of acetonitrile, 1 ml triperoxonane acid; 0-100%). Integrating chromatographic analysis gives purity above 95%.

N-terminal sequence of amino acids

N-terminal amino acid sequence is determined using AB1 470 - sequencing machine with a standard linear program and PTH-detection. Found a sequence of ST-Y2-Q3-G4-N5-S6-D7-C8-Y9 coincides with the expected originating from a DNA sequence.

r die gesamte innere Medizin" (ZGIMAL), 42, (17) 478 - 486 (1987). Specific activity is of 550,000/mg,001/mg. Stimuliruemoe K2P from E. coli Enrichment fragments of fibrinogen (activity in the presence of fibrinogenic fragments attributed to activity without fibrinogenic fragments) in this test system is not more than 25.

C) Binding to fibrin in vitro

In-vitro binding of K2P from E. coli with fibrin determined as described (Higgins, D. L. und Vehar, G. A. (1987), Biochem, 26, 7786-7791).

In Fig. 2 shows that K2P from E. coli as opposed to the full t-PA on CHO or t-PA from E. coli does not possess any appreciable ability to bind fibrin.

Example 6. The increase of expression level.

To increase the yield of the target product coding K2P gene sequence perekodiruya in a plasmid with a higher number of copies. For this purpose the plasmid pePa 126.1, described in the patent Germany 3838378.0. This plasmid consists of the vector pKK223-3 and the coding sequence for t-PA, as described in European patent A-O 242 835.

In this plasmid primarily integrate the sequence fd-terminator. For this purpose, the plasmid pePA 126.1 linearizer with restriction enzyme Hind III. Split thus Gentz and other (1981), PNAS 78(8): 4963) is subjected to restriction with Hind III and preparative get the fragment Hind III size is about 360 p. O. that contains fd-terminator (by gel electrophoresis and gel elution). The linearized plasmid pePa 126.1 and Hind III fragment from pLBU1 length 360 p. O. are ligated. The mixture after ligating contractorised described in the patent application Germany 3838378.0 the plasmid 500 pUBS in E. coli DSM 2102. From clones to choose those that contain the desired plasmid pePA 126 fd, which differs from the original plasmid pePA 126.1 fact that it contains a second site cutting Hind III.

From a plasmid pePA 126 fd receive two fragments:

BamHI/PvuI fragment size 3,4, etc., O. and PvuI/XmaI fragment size of about 1.3, etc., acting Both these fragment are ligated with 1.3 T. p. O. BamHI/XmaI fragment from plasmid pA27.3 and transforming E. coli with plasmid pVBS 500. The resulting plasmid name pA 27 fd.

Example 7. Preparation of pharmaceutical compositions.

Derived from E. coli t-PA - derived K2P after the above purification can be used for thrombolytic therapy, preferably in the form of pharmaceutical compositions.

Appropriate pharmaceutical drugs in particular may have the following composition:

0,05-02 mol/l tween 80

3. 0.1 to 0.2 mol/l phosphate of sodium or potassium, pH 6,0

10 mol/l tranexamic acid,

50 mg/ml sucrose or trehalose and

0.01 to 0.2 wt.% tween 80 or tween 20.

For K2P from example 3, the concentration of protein in any of the above compositions 4 to 10 mg/ml (amidopirina activity of 2,4-4.9m/ml).

Example 8. Pharmacological data obtained in prokaryotes K2P.

1. Pharmacokinetics K2P rabbits

K2P white new Zealand rabbits compared with Actilyse in their pharmacokinetic properties. Both fibrinolytic injected at a dose of 200 000 IU/ kg KG 30 min plasma Samples get at certain points in time before, during, and after infusion. t-PA activity was measured using the spectrophotometric test J. H. Verheijen and others (Thromb. Haemostas. 48, 266) (1982)) and modified by H. Lill (Z. ges Inn. Med. 42, 478 (187)).

For calculation of pharmacokinetic parameters calculated using the nonlinear regression, modified by H. J. Huang (Aero-Astronautica-Report 64. Rice University, 1 - 30, 1969). The parameters calculated individually using biexponentially pharmacokinetic model.

K2P shows five times longer half-life (t1/2 = 10,3 min, reducing concentred) in the case of K2P Pro concentration in the plasma activity of t-PA (CINF./equal 1966 1 U/ml and, thus, six times higher than in the case of Actilyse . The distribution volume (Vc) is for K2P 46,8 ml/kg compared with 73,7 ml/kg for Actilyse. The clearance of the entire plasma (C1totalcompared with Actilyse Actilyse(C1total= 22,2 ml/min/kg) in the case of K2P Pro reduced to 3.2 ml/min/kg When applied fibronolytic especially interesting the AUC, as it reflects the primary concentration in plasma. K2P shows AUC 8 times higher (1064 1/MLC) than Actilyse Actilyse(133,3 1U/MLC).

In General, K2P compared with Actilyse(it's currently only selling recombinant t-PA protein) with an equal dose shows improved 5 - 8 times the pharmacokinetic profile.

2. Pharmacodynamics K2P rabbits

For trials of thrombolytic use efficiency developed by D. Collen, and other model in rabbits by thrombosis of the jugular vein (J. Clin Invtst. 71,368, 1983). K2P and Actilyse Actilyseexplore three, depending on the circumstances, doses. Fibrinolitiki pour in for 4 h and then determine the speed of thrombolite (PL. 7, Fig. 5).

Calculated using linear extent regression dose for 50% thrombolite (ED50) is for K2P 124000 1Uctilyse. K2P provides, depending on the dose, the concentration activity of t-Pa in plasma which, when reduced fourfold dose comparable with Actilyse. Comparable thrombolytic action with 800 kU ActilyseKG dose of 200 kU K2P/ kg KG leads to slight changes in acquisition parameters fibrinogen, plasminogen and2-antiplasmin that it does not differ from the effects of a dose of 800 to U Actilyse /kg KG.

K2P represents t-PA-Mutein, which on the model of thrombosis of the jugular vein of a rabbit during a four-hour infusion at a dose of constituting a 1/4 dose Actilyseshows the same thrombolytic effect as ActilyseK2P not differ at low dose of Actilysethe effects on the clotting system and the concentration in plasma t-PA activity.

Example 9. Pharmacological properties of K2P from E. coli to be used as a model dog with thrombosis of the coronary artery

In order to study the thrombolytic action of K2P from E. coli on blood clots experimental model was chosen animal with acute myocardial infarction. As one of these animals explored the dog. The method of formation of a blood clot crown is ahogadas under General anesthesia and artificial respiration dogs irritate the intimal surface of the surrounding branches of A. coronaria sinistra (=leftcirtum - flex coronary artery = LCX) electric pulse (150 ) thus occurs thrombosis.

While proximally coronary thrombosis received by means of electromagnetic floating measuring head, providing the ability to measure reperfusion (restoration of blood flow).

To establish the necessary doses under the action of heparin dogs for 1 min was administered once by intravenous injection of BM 06.022 with eucharistically t-Pa(Alteplase, Actilyse, Dr. Karl Thomae GmbH. Biberoch, Germany) in the form of four different doses (6 animals per dose). Before and at specific time points after injection were selected plasma samples to determine the concentration of plasma t-PA activity, fibrinogen, plasminogen and2-antiplasmin, as well as to determine the number of platelets in the whole blood. Fibrinogen was measured coagulopathies by Klaus (Acta haemat. 17, 237, 1957), plasmagun and2-doesn? t measure, as described by Collen and others (J. Clin Invest 71,368, (1983) spectrometrically.

Next Simplate bleeding time was measured on the rear leg of the dog with the latch (SimplateI, Organon I, Organon Teknika, Eppelheim, Germany) at 40 mm Hg (J. Surg. Res. 27, 244, 1979). Static comparison of the measured values after injection with the control value before the injection is performed using Wilcoxon test for each pair.

50). Statistical comparison of the weight of residual thrombus is performed using Wilcoxon test Mann-Whithey.

Concentration in Plaza t-PA activity was measured using the spectrophotometric test Verhejen and others (Thromb Haemost. 48, 266, 1982), modified by Lill (z gesamte Inn Med. 42, 478 (1987). For calculation of pharmacokinetic parameters is used the estimated nonlinear regression, modified by HY. Huang (Aero-Astronauties Report 64 Rice Univercity, USA, 1 - 30, 1969). The parameters are calculated after reducing the inherent basal level of t-PA activity following measured values using biexponentially pharmacokinetic model.

When this is obtained the following results:

1. Farmakodinamika on dogis 1600 U/kg KG. Comparing the value of the ED50for K2P Actilyseshows that the first (83 U/kg KG) approximately 11.5 times lower than the second (961 U/kg KG). The introduction of a placebo does not restore blood flow. The weight of residual thrombus introduced with placebo is 9.6 1.6 kg (mean SEM; K2P, and also Actilyselead with increasing doses to a statistically significant reduction in the weight of residual thrombus compared with placebo control. Reperfusion occurs both fibrinolitikov average in all animals after 25,9 3.5 minutes (K2P) and later 24,2 6.2 min (Actilyse Actilyse).

The majority of treated K2P or Actilysedogs re occluder after recovery (table.8).

2. Pharmacokinetics in the dog

After intravenous injection of 200 U/kg K2P or Actilysein the anesthetized dog shown that the rapid phase of decrease in plasma concentrations, expressed as t 1/2 , in the case of K2P (7,2 1,1 min) 4:5 times longer than in the case of Actilyse(1,6 0,2 min) (table.9). Found immediately after the injection, the plasma concentration K2P approximately twice higher than in the case of Actilyseilyse. Accordingly, the area under the curve of plasma concentration K2P about 9.5 times more than that in Actilyse.

3. Specificity for fibrin on the dog

After 2 h after injection K2P find in a dose-dependent small reduction in residual concentration of fibrinogen (10 to 81% in the case of the highest dose of 200 U/kg KG). In contrast, fibrinogen concentration after administration of the highest dose (1600 U/kg KG almost completely reduced to 3 0% (table. 10). If you carry a semi-log regression analysis of dependence "dose - side effect (reduction ratio of fibrin) and determine the ED50thrombolytic action against residual concentration of fibrinogen, for equipotent doses in the case of K2P residual content of fibrinogen obtained 92,5% (compared to the 38.6% in the case of Actilyse). For plasminogen and2-antiplasmin are also dose-dependent decrease of the residual content after 2 h after injection, which in the case of Actilysemore pronounced than in the case of K2P. Only the concentration of platelets in the case of both substances are equally unaffected.

4. Effect on bleeding time in dogs

Intravenous is determined as being a value before injection in all four different doses. In contrast, Actilysein doses 1130 and 1600 U/kg KG statistically significantly prolong bleeding time (Fig.6 and 7).

5. The overall score

On the described model of thrombosis of the coronary arteries K2P manifests itself as a thrombolytic, which after intravenous injection portion may reach 100-Noah speed of restoration of blood flow without affecting much on the concentration of fibrinogen and without appreciable elongation bleeding time. Compared with ActilyseK2P in the thrombolytic potency after intravenous injection was clearly superior (factor of 11.5). Study the pharmacokinetic profile K2P also shows that compared with Actilyseclearance K2P as the expression for a longer removal from plasma, nine times longer.

1. The DNA fragment encoding the polypeptide KR properties of plasminogen activator having the nucleotide sequence represented on the page. xxx containing ATS of the start codon.

2. Polypeptide KR properties of plasminogen activator obtained by transformation of the bacterial strain with the recombinant vector comprising the DNA fragment with the sequence described in paragraph 1 or similar which you want to make of 550,000-ed/mg; molecular weight 38500 2000 Yes; increased half-life compared to the natural counterpart; fiberisima stimulation; thrombolytic activity; solubility in the solution of arginine at a concentration of 10 to 1000 mmol/l with product purity of 95%.

3. Recombinant plasmid DNA RA.3 encoding a polypeptide KR properties of plasminogen activator containing EcoR I - EcoR I fragment of DNA plasmids pREM 7685; TGTCT TAC CAA GGA AAC AGT GA - synthetic DNA fragment; xhoI - xhoI - fragment DNA plasmids pREM 7685; three sites of cleavage of restrictase ECOR I; one site of cleavage of restrictase xho I; tac - promoter, genetic markers; Apr- gene resistance to ampicillin; Kmr- gene resistance to kanamycin.

4. Pharmaceutical composition having fibrinolytic activity, containing a plasminogen activator and a pharmaceutically acceptable carrier, characterized in that as plasminogen activator it contains the polypeptide CR under item 2 in an effective amount.

 

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The invention relates to medicine

FIELD: medicine, biotechnology, pharmaceutical industry, veterinary science.

SUBSTANCE: method involves addition of tris-HCl buffer (pH 7.5) containing 0.1 M of lysine and 5 mM of EDTA to the Cohn's fraction followed by incubation and centrifugation. Prepared suspension is incubated with 10% PEG-3000 and after centrifugation the solution is applied onto column with lysine-Sepharose at pH 7.5 and plasminogen is eluted with buffer solution at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine and 5 mM of caprylic acid. Then plasminogen is incubated with streptokinase in tris-buffer (pH 7.5) containing 15 mM of lysine, 10 mM of ε-aminocaproic acid and 50% of glycerol. Then method involves chromatography on column with benzamidine-Sepharose at pH 8.5 and elution with buffer at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine, and the end product is lyophilized that comprises about 30 IU/ml of plasmin with purity above 98%, at least 10 mM of lysine and 10 mM of glycine in an aqueous solution at pH about 3.5. The prepared product shows apyrogenic property, absence of toxicity in experiments in laboratory animals and it doesn't cause allergic or other adverse response reactions in intracutaneous or intravenous administrations, and the preparation doesn't cause defects in the vision field after its an intraorbital route of administration.

EFFECT: improved preparing method, valuable medicinal properties of plasmin.

7 cl, 1 ex

FIELD: biology, biochemistry, science-practical pharmacology, molecular biology.

SUBSTANCE: invention relates to isolated DNA fragment with indicated nucleotide sequence encoding protein that elicits property of intramembrane endoprotease (IMPAS), and to a method for preparing this protein. Invention provides the possibility for development of preparations used for diagnosis of diseases associated with proteolysis of intracellular receptors. Invention provides expanding assortment of agents useful in treatment of different neurological, cardiovascular and other diseases associated with the disturbed proteolysis of proteins.

EFFECT: improved preparing method, valuable medicinal properties of protein.

3 cl, 4 dwg, 1 tbl, 3 ex

FIELD: medicine, pharmaceutics, cosmetology.

SUBSTANCE: the present innovation deals with medicinal preparations and application of fish serine proteinases chosen out of the group consisting of trypsin, chemotrypsin and their any mixture obtained out of cod, or having, at least, 90% homology according to amino acid sequence with trypsin or chemotrypsin isolated out of Atlantic cod, as medicinal preparation for treating and/or preventing diseases where receptor-mediated binding is involved into pathogenesis. Also, it is suggested to apply pharmaceutical composition for treating and/or preventing both human and animal disease where, also, receptor-mediated binding is involved and that contains efficient quantity of serine proteinase mentioned and pharmaceutically acceptable solvent or carrier, and cosmetic composition for removing dead or desquamative skin from healthy skin that contains efficient quantity of fish serine proteinase mentioned. The innovation provides application of serine proteinases both for medicinal and cosmetic objectives.

EFFECT: higher efficiency of application.

24 cl, 4 dwg, 14 ex, 6 tbl

FIELD: biotechnology, biochemistry, medicine, veterinary science.

SUBSTANCE: method involves preparing a proteolytric enzyme solution being protosubtilin G3H, G10H or G20H mainly in 0.05 M Na-phosphate buffer, pH 7.2-8.2. Prepared solution is purified by saline precipitation of inert proteins by sequential addition of calcium chloride and sodium phosphate followed by keeping the mixture at 8-10°C for 16-18 h and filtration of precipitated deposit through paper and membrane filters. Prepared filtrate is desalted additionally, concentrated and purified by ultrafiltration in hemodialysis columns. The purification process is carried out for two stages by dilution of concentrate with distilled water up to the parent volume and the following repeated concentrating. Polyethylene oxide of molecular mass 1500-4000 Da is added to the prepared highly purified preparation of proteases up to the ratio enzyme - carrier = 1:(3-8). The mixture enzyme - carrier is subjected for repeated sterilizing filtration through membrane filter and irradiated by bremsstrahlung (braking) γ-radiation in the dose 0.5-1.0 Mrad. Invention provides preparing the sterile apyrogenic solution of proteases immobilized on polyethylene oxide with the proteolytic activity 80-120 PU/ml. Invention can be used in preparing injection preparations of immobilized enzymes.

EFFECT: improved preparing method.

4 cl, 1 tbl, 5 ex

FIELD: veterinary science, animal feeding.

SUBSTANCE: the suggested composition contains esterase-phospholipase or hemicellulase and a physiologically acceptable carrier. The method for treating and decreasing the risk of digestive tract infection deals with peroral introduction of the mentioned enzymes or application of enzymes or peroral medicinal forms of enzymes as a preparation for treating digestive tract diseases.

EFFECT: higher efficiency.

72 cl, 2 dwg, 15 ex, 20 tbl

FIELD: medicine, pharmaceutical industry, phytotherapy.

SUBSTANCE: invention proposes an agent used in treatment of infectious diseases. Agent used in treatment of infectious diseases comprises chitosan of polyfraction composition with amine groups of molecular mass from monomeric link - glucosamine to ˜350 kDa and deacetylation degree from ˜68 to ≤ 95% and vegetable raw aqueous extract chosen from the following group: Cetraria thallus, sage official herb, cocklebur leaves, black poplar buds, common wormwood herb, red roots, burdock roots, French honey-suckle herb, bur-marigold tripartite herb, sandy common immortelle flowers, dropwort elm-leaved flowers, river avens (Geum) roots, licorice glabrous roots, swampy sweet flag roots, burnet official roots,, nettle dioecious herb, swampy cudweed herb, common milfoil herb, sweet clover official herb, gum-free leaves, dropwort, willow bark, birch leaves, bilberry leaves, willow-herb narrow-leaves herb, peppermint herb, fenestrate Saint-John's-wort herb, knot-grass herb, shepherd's purse herb, violet pensy tricolor herb, speedwell herb, pot-marigold flowers, common tansy flowers, matricary official flowers, chicory roots, dandelion official roots, tormentil roots, cinnamon wild rose fruits, viburnum leaves, pinnate kalanchoe and maize stigmas. Aqueous extract is prepared by extraction of vegetable raw with demineralized water under definite conditions. Agent promotes to effective treatment of infectious diseases, such as chlamydiosis, herpes, erysipelas, viral hepatitis A and B, gastroenteric tract diseases, acute enteric infectious, and to declining period of clinical symptoms of diseases said.

EFFECT: valuable medicinal properties of agent.

10 cl, 2 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: method involves carrying out Indomethacin and Chondroxid ointments phonophoresis with enzymatic agent of Caripazyme. Indomethacin and Chondroxid are mixed in 1:1 proportion and Caripazyme is added in the amount of 50 mg dissolved in 5 ml of physiologic saline.

EFFECT: improved synergetic interaction of drug and physical factor.

4 dwg, 1 tbl

FIELD: medicine, dermatology, in particular treatment of trophic ulcer (TU) and long-term open septic wound (LTOSW).

SUBSTANCE: claimed method includes application of wound-healthing composition onto TU and LTOSW in the first step of wound process. Said composition contains (mass %): methyluracil 0.9-1.1; pepsin 3.4-4.4; bentaine hydrochloride 3.6-4.4; sodium chloride 9.0-11.0; and balance up to 100,0: distilled water. In the second and third phases fine cut collagen preparations in form of 3-4 mm thickness layer are applied on purified TU and LTOSW surface. Then cotton carrier freely moistened with wound-healthing composition diluted with water in ratio of 1:2 is layered onto collagen layer. Multihole microirrigator and further sterile cotton carrier are sequentially applied over abovementioned cotton carrier.

EFFECT: effective treatment due to accelerated abruption of necrotic tissues, inhibition of microbial growth, decreased risk of infective, toxic and allergic affects, and improved tissue regeneration.

2 ex, 3 cl

FIELD: biotechnology, medicine.

SUBSTANCE: claimed conjugate contains fibrin/fibrinogen-binding fragment bound to pharmaceutically active agent directly or via intermediate fragment trapping said agent, such as antibody. Conjugate may represent recombinant fused protein comprising C-terminal domain of VEGF165, fused with substance for wound healing, such as leptin.

EFFECT: conjugate capable of depot formation for prolonged elimination of pharmaceutically active agent from fibrin gout.

15 cl, 13 dwg, 8 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention proposes a composition comprising one or more corticosteroids and/or hormonal steroids in the range from 0.025 to 5 wt.-% of the total composition mass and hyaluronidase in the amount from 25 to 4000 UTR per 1 g of the composition in the suitable carrier medium. The composition can comprise nonhormonal anti-inflammatory agents. The composition can be prepared in different medicinal formulations, such as gel, ointment, cream, aerosol with additives known from a person skilled in the art of the corresponding field. Betamethasone is the preferable corticosteroid. Ointment is the preferable medicinal formulation. Treatment by using corticosteroids in combination with hyaluronidase provides the improvement of state in 90% of patients, and after their treatment their prepuce can be drawn off easily.

EFFECT: improved medicinal properties of composition, enhanced effectiveness of treatment.

11 cl, 6 ex

FIELD: organic chemistry, pharmacy.

SUBSTANCE: invention relates to an improved chelate conjugates of the formula: wherein each R1, R2 and R3 represents independently R group; Y represents -(A)n-X-Z wherein X represents -NH-, -CO2-, -N(C=O)-; Z represents a biological group for directed delivery that is chosen from 3-20-membered peptide, substrate for enzyme, enzyme inhibitor or synthetic compound binding with receptor; -(A)n represent linker wherein each A represents independently -CR2-, -NRCO-, -CONR-, -CR2OCR2 or polyalkylene glycol group; n represents a whole number from 2 to 10; each R group represents independently hydrogen atom (H) or (C1-C10)-alkyl, or two R groups in common with atoms to which they are added form (C3-C6)-carbocyclic saturated ring. Complexes with radioactive metals are useful as radioactive pharmaceutical preparation, especially, with 99mTc.

EFFECT: valuable medicinal properties of conjugates.

30 cl, 3 tbl, 4 dwg, 30 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention provides novel compounds of general formula (I) and pharmaceutically acceptable salts thereof, wherein G represents glycin; D aspartic acid; R1 group -(CH2)n- or -(CH2)n-C6H4-; n is integer from 1 to 10; h is 1 or 2; X1 represents amino acid residue having one functional deviation such as amine; X2 and X4 independently represent amino acid residue capable of forming disulfide bond; X3 represents hydrophobic amino acid such as phenylalanine; X6 represents group -NH-[CH-]-C(O)-; X7 is missing or represents biomodifying grouping consisting of monodisperse poly(ethylene glycol): (II), wherein is integer from 1 to 10; C-end unit represents amide group or 1-10 amino acid residues; Z1 chelating group or reporter group; and W1 is missing or represents spacing moiety derived from glutaric or succinic acid. Compounds of invention are designed as diagnostic agents for visualization or as therapeutic agents, the two agent types being directional vectors capable of binding to integrin receptors.

EFFECT: expanded diagnostic and therapeutic possibilities.

19 cl, 3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: various polypeptide of VII human coagulation factor characterized by high coagulating activity in comparison to natural factor VIIa and not requiring tissue factor induction for development. Structurally new factors VII are amino acid sequence of natural protein containing substitute L305 Val/Tyr/Ile and one or more substitutes including K337 Ala/Gly/Val/Ser//Thr/Asn/Gln/Asp/Glu; V158 Ser/Thr/Asn/Gln/Asp/Glu; E296 Arg/Lys/Val and M298 Lys/Arg/Gln/Asn. Genetic make-up and expression systems for production of described various factor VII by means of recombinant DNA technologies, as well as application of new polypeptides composing pharmaceutical formulations.

EFFECT: compounds are used for bleeding treatment and coagulation system upkeeping.

31 cl, 1 dwg, 7 ex

FIELD: gene engineering.

SUBSTANCE: invention is related to protein and gene engineering and can be used in pharmaceutical industry. New options of hemostasis factor VII are proposed with high proteolytic activity as a result of replacement of amino-acid residue positioned in natural sequence as Met 298 human factor VII i.e. glutamine residue. According to invention, factor VII may include additional replacements in some other positions of proteinase domain and be represented with multi-sited mutant forms, particularly [V158T/M298Q]F-VIIa, [V158D/M298Q]F-VIIa, [V158D/E296V/M298Q]F-VIIa and [V158D/E296V/M298Q/K337A]F-VIIa. New options of factor VII are produced by recombinant DNA method. To implement this method coding nucleotide sequences, vector structures and transformed cells are proposed.

EFFECT: invention allows to use new mutant forms of factor VII for production of medicines with coagulant activity.

20 cl, 1 dwg, 2 tbl, 13 ex

FIELD: medicine, biotechnologies.

SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.

EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.

20 cl, 2 dwg, 8 ex

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