HK1010109B - Bifunctional derivatives of urokinase with improved fibrinolytic activity and thrombin inhibiting activity - Google Patents

Description

The invention relates to bifunctional urokinase variants with enhanced fibrinolytic properties and thrombin-inhibiting effect, plasmids for use in the production of these polypeptides and thrombolytics containing a bifunctional urokinase variant as an active substance.

A key feature of human blood is its ability to close up blood vessel injuries by forming blood clots. Blood clotting is caused by a series of enzymes in the blood, which in the so-called clotting cascade ultimately cause the enzyme thrombin to proteolytically convert the precursor protein fibrinogen to fibrin. Fibrin polymerizes to include platelets, erythrocytes and other blood components at the site of the lesion to form a thrombus.

In addition, blood contains a number of enzymes that prevent clotting and ensure blood flow after the vascular walls regenerate. The most important enzyme for thrombolysis is plasmin, which proteolytically attacks the fibrin spindle and thereby causes the dissolving of the thrombus. Plasmin is formed by proteolytic cleavage of the inactive precursor protein plasminogen. Activation is caused by plasminogen activators by proteolytic cleavage of the plasminogen.

Heart attack and stroke are closely linked to the pathological formation of thrombosis. In both forms of stroke, thrombosis occurs in the blood vessels, usually as a result of arteriosclerotic changes in the arteries. These thromboses disrupt blood flow in the arteries, so that the tissue can no longer be adequately supplied with oxygen. This leads to partial or complete death of the heart muscle in heart attack.

Plasminogen activators are used as thrombolytics to initiate the breakdown of platelets by plasmin in patients with heart attacks. Currently, therapies include streptokinase, APSAC (Anisolated Plasminogen Streptokinase Activator Complex), bi-directional urokinase (UK), recombinant single-chain urokinase (recombinant prourokinase) and tissue plasminogen activator (tPA) (Collen and Linen, Blood 78, 3114 - 3124 (1991)). Streptokinase is a protein that modifies streptokinase. Streptokinase activates plasminogen by forming a complex with plasminogen and thereby converting the plasminogen into an active conformation. This complex is then released into the plasma plasma to re-activate the active plasminogen and then into the plasma. APSAC is a biologically active compound of plasminogen and is a modification of the plasma.

Urokinase is a human protein that can be obtained from urine in two forms as a proteolytically active protein: high-molecular urokinase (HUK) and low-molecular urokinase (LUK) (Stump et al., J. Biol. Chem.261, 1267 - 1273 (1986)). HUK and LUK are double-chain molecules. Urokinase is formed as a monoclonal urokinase (prourokinase) in various tissues and can be detected as a proenzyme in small amounts in human blood (Wun et al., J. Biol. Chem. 257, 3276 - 3283 (1982)).Although prourokinase and plasminogen are present as proenzymes, prourokinase is capable of converting plasminogen into active plasmin due to intrinsic activity, but this plasminogen activator does not become fully active until the plasminogen formed has in turn split the prourokinase between 158Lysin and 159Isoleucin (Lijnen et al., J. Biol. Chem. 261, 1253 - 1258 (1986)). The genetic gain of urokinase in Escherichia coli was first described by Heynckes (Proceedings of the International Symposium on the Genetics of Microorganisms 1982).Unglycosylated prourokinase (saruplase) is produced by the use of a synthetic gene (Brigelius-Flohé et al., Appl. Microbiology.

Tissue plasminogen activator is a protein found in blood and tissue with a molecular weight of 72 kilodaltons. This plasminogen activator consists of 5 domains: the amino-terminal finger domain, the growth factor domain, the kringel 1, the kringel 2, and the serine protease domain. Unlike prourokinase, tPA is only able to break down plasminogens by binding to fibrin. Like prourokinase, tPA is transferred to the active form by a plasma-catalyzed cleavage between kringel 2 and the serine protease domain.

Since the early 1980s, active treatment of myocardial infarction with thrombolytics has been shown to be effective and efficient. A number of studies have shown that the treatment of heart attack patients with streptokinase, APSAC, UK, recombinant prourokinase or tPA leads to a significantly reduced mortality compared to untreated patients. To improve the efficacy of these substances in therapy, a number of derivatives of the tissue plasminogen activator and prourokinase have been produced using genetic methods. In addition to increased fibrinolytic activity and reduction of side effects, the development of forms suitable for bolus application is at the centre of the study. An overview of approaches to enhancing plasminogen activator and thrombosis is contained in Haemostasis (1996, 86 and 90), and in the Trost 90 (1996, 88 and 9), and in Biotechnology (1991, 88 and 110).

To improve the efficacy of plasminogen activators in lysotherapy and in particular to increase their biological half-lives, deletion and substitution variants of the tissue plasminogen activator have been developed, for example by removing the finger and growth factor domains or by exchanging the serine protease domains for the serine protease domains of urokinase (Collen et al., Thromb. haemostasis 65, 174 - 180 (1991); Fromage et al., Fibrinol 5, 187 - 1901); Blood et al., Blood 78, 125 - 131 (1991)). In fact, the original deletion of the Fibrinol and growth factor variants showed that the biological value of the plasma t-activator was significantly increased (Fibrinol and Fibrinol, 78, 94 - 94), but this was not the case due to the length of the plasma t-activator (Plasminol and Fibrinol, 78, 94 - 95), which was a long-acting plasma and a small amount of blood plasma (Fibrinol and L-glucan, 131 - 95).

For example, a variant of tPA is known where kringel 1 is replaced by kringel 2 of the parent molecule. This variant has an increased affinity for N-terminal lysine residues but not for fibrin. In animal models, this variant was not more reactive to thrombolysis than the original plasma plasma activator (Lifer et al. Haemoglobinase 6., 1801); other known plasma activators are Fibromycin et al. Fibromycin and Fibromycin (Glyphosate, 174, 174, 224, 224, 224, 264, 264, and 264, and 224, and 224, and 264, respectively).

However, the success of treatment of stroke patients with plasminogen activators depends not only on thrombolysis but also on the extent to which the re-closure of opened blood vessels can be prevented. Various findings indicate that thrombin bound in the blood vessels is released back into the blood as an active enzyme during thrombolysis and can cause re-closure of vessels (Szczeklik et al., Arterioscl. Thromb. 12, 548-553 (1992); Eisenberg, Circulation 84, 2601-2603 (1991)). In fact, the effect of thrombolytic therapy is significantly improved by simultaneous or pre-existing reduction of the thrombolin group.

One of the most potent thrombin inhibitors is hirudin, isolated from the leech Hirudo medicinales, which binds specifically to the so-called anion binding site of thrombin with its carboxy terminal half. Certain amino acids of the amino terminal half of the hirudin molecule block the access to the thrombin substrate binding pocket (Rydel et al., Science 249, 277-280 (1990)).

The use of hirudin in combination with a plasminogen activator for the treatment of thrombotic vascular disease is described in European patent applications EP 328 957 and EP 365 468.

International patent application WO 92/18139 describes chimeric molecules with a plasminogen activating component not bound to fibrin and a component with an affinity for non-fibrin components of arteriosclerotic plaques, such as a thrombin inhibiting component. The two components may be linked by a 12-mer consisting of alanine or containing predominantly alamine. Within an alanine-rich 12-mer, 2 or 3 alanine residues may be replaced by lysine or arginine.

International patent application WO 91/09125 identifies fusion proteins with a cleavage point between the first and second sequence which have a fibrinolytic and/or antithrombotic effect. The full fibrinolytic and/or antithrombotic effect is achieved only by cleavage, which can be produced in particular by thrombin.

Thrombin can also be inhibited by a peptide derived from the amino-terminal sequence of the human thrombin receptor (Vu et al., Nature 253, 674-677 (1991)). The thrombin receptor contains a thrombin-binding sequence with an adjacent cleavage site for thrombin in the amino-terminal region. The thrombin-binding region of the receptor is very similar in structure to the carboxy-terminal region of hirudine. The receptor is activated by thrombin by cleavage of the receptor sequence.

Similarly, thrombin can be inhibited by a peptide derived from the amino acids 41 to 57 of haemadin (Strube et al., J. Biol. Chem. 268, 8590-8595 (1993).

The purpose of the invention was to develop active substances for the treatment of thrombotic vascular occlusion which would produce complete thrombolysis within a very short time while preventing re-closure of the vessels after initially successful thrombolysis.

It has now been found that certain bifunctional urokinase variants meet the high requirements for such active substances.

The invention is therefore concerned with bifunctional urokinase variants of the general formula I. M4-X1-Y1 In which M4the amino acid sequence 47Ser to 411Leu of the unglycosylated prourokinase as shown in Figure 1 (SEQ ID NO: 21 and 22) means that X1 is a direct link between M4 and Y1, or a peptide of the sequence The following shall be added to the list of substances: or The following shall be added to the list of substances which are to be classified in Annex I: or The following shall be added to the list of substances which are to be classified as substances of heading 2913: or other peptide sequence of general formula II The following shall be reported for the following categories of vehicles: where X2 Pro or Leu, X3 Val or Pro, X4 Lys, Val, Arg, Gly or Glu, X5 Ala, Val, Gly, Leu or Ile, X6 Phe, Trp, Tyr or Val and X7 Gly or a direct link between X6 and Y1 means, andY1 is a peptide of the sequence The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council: or The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council: or The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: is Y2 Pro or Val, Y3 Leu or a direct bond between Pro and Gly and Y4 Gln or a hydroxyl group.

In bifunctional urokinase variants of the general formula I, where Y1 is a peptide of the sequence The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council: wherein Y2 Pro or Val, Y3 Leu or a direct bond between Pro and Gly and Y4 Gln or a hydroxyl group is represented, X1 is preferably a peptide sequence of general formula II The following shall be reported for the following categories of vehicles: wherein X2 Pro or Leu, X3 Val, X4 Lys, Val or Arg, X5 Ala, Val or Gly, X6 Phe, Trp, Tyr or Val and X7 Gly or a direct binding between X6 and Y1. In particular, preference is given to peptide sequences of general formula II in these bifunctional urokinase variants wherein X2 Pro or Leu, X3 Val, X4 Lys or Val, X5 Ala or Val, X6 Phe, Trp or Tyr and X7, Gly or a direct binding between X6 and Y1 and in particular wherein X7 means a direct binding between X6 and Y1.

In bifunctional urokinase variants of general formula I, where Y1 is a peptide of the sequence The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council: represented by Y2 Pro or Val, X1 is preferably a peptide sequence of general formula II The following shall be reported for the following categories of vehicles: wherein X2 Pro or Leu, X3 Val, X4 Lys or Val, X5 Ala or Val, X6 Phe or Trp and X7 are a direct link between X6 and Y1.

Compared with known plasminogen activators and known mixtures of a plasminogen activator and a thrombinine inhibitor, the bifunctional urokinase variants of the invention are characterised by a stronger fibrinolytic effect, combined with unpredictably good thrombinine inhibitory properties. In addition, the polypeptides of the invention surprisingly consume plasma fibrinogen in significantly lower amounts. The resulting significantly higher fibrin specificity, especially also in the known mixtures of a plasminogen activator and a thrombinine inhibitor, means that the blood clotting capacity is affected only slightly and the risk of uncontrolled blood clotting as a possible complication of a possible thrombinase system is significantly reduced compared to the use of bolus-like urokinase.

Bifunctional urokinase variants of generic formula I are toxicologically safe and therefore can be administered as such in appropriate pharmaceutical formulations to patients with thrombotic vascular occlusion.

The other subject matter of the invention is therefore thrombolytics containing as active substance a bifunctional urokinase variant of the general formula I.

The treatment of thrombotic vascular occlusion, such as heart attack, cerebral infarction, peripheral acute arterial occlusion, pulmonary embolism and deep leg and pelvic vein thrombosis requires 0.1-1 mg/kg of a polypeptide of the invention.

The thrombolytics of the invention contain, in addition to at least one bifunctional urokinase variant, excipients such as carriers, solvents, diluents, dyes and binders, the choice of which excipients and the quantities to be used depend on the method of administration and do not cause any problems for the practitioner.

The bifunctional urokinase variants are produced by genetic engineering, and the invention also covers plasmids for use in the production of bifunctional urokinase variants of general formula I, the operon of which has a regulable promoter, a shine-dalgarno sequence acting as a ribosome binding site, a start codon, a synthetic structural gene for a bifunctional urokinase variant of general formula I and one or two terminators downstream of the structural gene.

The trp-promoter or tac-promoter is particularly suitable as an adjustable promoter, and the trp A terminator and/or the tet A/or L terminator from Tn 10 are preferably used as terminators.

In the control region of the plasmids of the invention, the distance between the Shine-Dalgarno sequence and the start codon is 6-12 nucleotides, preferably 8-10.

The expression of the plasmids of the invention is carried out in Escherichia coli strains, in particular in Escherichia coli strains of group K 12, e.g. E.coli K 12 JM 101 (ATCC 33876), E.coli K 12 JM 103 (ATCC 39403), E.coli K 12 JM 105 (DSM 4162) and E.coli K 12 DH 1 (ATCC 33849). In the bacterial cell, the bifunctional urokinase variants of general formula I of the invention occur in high doses in isolates containing the protein in its isolated form. After isolating the isolates, the saturated protein is converted into a protein in the tertiary structure by the reactive chemistry of a redox system.

Examples 1) Identification, isolation and purification of bifunctional urokinase variants according to the invention (a) Cloning work

The expression plasmids for the genetic engineering of the polypeptides of the invention in Escherichia coli were produced in a known way. The sequence of the individual manufacturing steps is shown in Figures 2 and 2a to 2p. The starting products of the plasmide production were the plasmids pBlueskript KS II+ (Stratagene, Heidelberg), pUC8 (Fa. Pharmacia, Freiburg) and pGR201 DNA. pGR201 DNA is DNA identification with the DNA described in EP 408 945 and Appl. Microbiol. Biotechnology 36, 640-649 (1992). The plasmids pBF160 are the restriction endonucleases BAMII, ClaodHI, Banco, NII, inhibitor, NII, Ndegenstein, NII and various modifying enzymes such as T-sequenziase (Gibasease-O4-Oxygenase) and T-sequenziase.

Err1:Expecting ',' delimiter: line 1 column 1255 (char 1254)

(b) Production of permanent crops and fermentation

Err1:Expecting ',' delimiter: line 1 column 382 (char 381)

The entire amount of culture obtained was then suspended in 1 l of standard I medium (pH 7.0; 150 mg/l ampicillin) and fermented in a shaker flask at 37°C. Induction was achieved by adding 2 ml of indolacrylic acid solution (60 mg in 2 ml ethanol) at an OD of 0.5 to 1 at 578 nm.

(c) Expression testing

To test the expression rate (plug units per OD per ml), cells corresponding to 1 ml of a cell suspension with an OD of 1 were centrifuged at 578 nm immediately before induction and every hour after induction (total of 6 hours). The centrifuged cells were opened with lysozyme (1 mg lysozyme per ml in 50 mM Tris/ HCl buffer, pH 8.0, 50 mM ethylene diammethylenetetracetic acid (EDTA) and 15 vol. % sucrose). The cells were dissolved in 4-5 m Guidinium hydrochloride solution and then, after dilution on Guidinium hydrochloride with the addition of a reducing agent (glutathione or Cysteine) for 2 to 2 hours, re-filled under 5 hours dilution (W. et al., 4045 of Biochemistry 2541 (1986)).The resulting single-chain bifunctional urokinase variants were converted by addition of plasmin to the corresponding bi-chain urokinase variants, whose activity was determined with the chromogenic substrate pyro-glu-gly-arg-p-nitroanilide, which is cleaved only by bi-chain active urokinases. The activation of the bi-functional urokinase variants of the invention with plasmin was achieved in 50 mM Tris/HCl buffer, 12 mM sodium chloride, 0.02 % Tween 80 at pH 7.4 and 37°C. The ratio of bi-functional urokinase variants to plasmin was approximately 100 - 1500 to 1 relative to molarity, or 8,000 - 36,000 relative to 1.Depending on the concentration of bifunctional urokinase variant, the reaction was stopped after 5 to 60 minutes incubation by addition of 50% acetic acid and extinction was measured at 405 nm. According to the manufacturer of the substrate (Kabi Vitrum, Sweden), this procedure results in an extinction change of 0.05 per minute at 405 nm at a urokinase activity of 25 Ploug units per test solution. The inventive bifunctional urokinase variants showed specific activity between 155,000 and 120,000 mg/ Ploug units per ml. The assay was performed on the BCA Pierce protein purification solution.

(d) Insulation and cleaning

After 5 to 6 hours of induction, the fermentation under the conditions described in 1b) was completed (density 5-6 OD at 578 nm). The cells were decentrifuged. The precipitate was resuspended in 200 ml of water and released in the high pressure homogeniser. After re-centrifugation, the precipitate containing the entire amount of the unkinetic bifunctional urokinase variant was dissolved in 500 ml of 5 M guanidinium hydrochloride, 40 mM cysteine, 1 mM EDTA at a pH of 8.0 and diluted with 2000 ml 25 mM Tris/HCl at a pH of 9.0. The reflux reaction was completed after approximately 12 hours.

The bifunctional urokinase variants were eluted with 0.5 M Tetramethylammonium Chloride (TMAC) in 0.1 M Acetate (pH 4) Buffer. After two chromatographic separations (copper chelate salicylate and cation exchanger), the urostachenous variants were obtained in pure form. N-terminal sequence analysis determined the variety and amino-terminal sequence of the desired amino acid. The protein change characterization of the carboxylate terminal was achieved by a 90 percent protein sequence analysis (CNP) of the individual carboxylate terminal after 1 ml of the peptide was removed from the CN-R protein (Peptoxypeptide) and the protein was removed from the peptide after 1 ml of the peptide was removed.

All bifunctional urokinase variants isolated and listed in Table 2 showed no or very low activity (below 1200 Plough units per mg purified protein) in a direct urokinase chromogenic substrate activity test. Only after cleavage with plasmin (conditions are given in section 1c) was enzyme activity between 120,000 and 155,000 Plough units per mg purified protein obtained. All urokinase variants were therefore expressed as single-chain proteins in E. coli K12 JM103. Other

(2) Pharmacological tests Determination of thrombin-inhibiting effect

The inhibitory activity of the bifunctional urokinase variants of the invention was determined by measuring the thrombin time by mixing 200 μl of a 1:10 dilution of human citrate plasma in veronal buffer with 50 μl of thrombin solution (0.2 units) and 50 μl of an aqueous solution containing 0.5 - 50 μg of a bifunctional urokinase variant. The time to formation of a fibrin clot was then measured. Table 3 lists the measured inhibitors indicating the prolongation of the thrombin time in the presence of 10 μg of each bifunctional urokinase variant of the invention. The concentration-dependent prolongation of the bifunctional time was also determined and for the bifunctional variants of urokinase M12, M12, M2, M2, M3, M3, M3, M3, M3, M3, M3, M3, M4, M4, and M4 was compared to the unprovoked pro-dosage of urokinase in accordance with the procedure for the formation of M4, M4, M4, and M4 (as opposed to the time to unprovoked by the invention of a bifunctional urokinase variant). Tabelle 3

bifunktionelle Urokinase-variante
M11 (SEQ ID NO: 23 und 24)	1,8
M12 (SEQ ID NO: 25 und 26)	4,6
M13 (SEQ ID NO: 27 und 28)	1,7
M14 (SEQ ID NO: 29 und 30)	1,8
M15 (SEQ ID NO: 31 und 32)	2,5
M16 (SEQ ID NO: 33 und 34)	3,2
M17 (SEQ ID NO: 35 und 36)	3,1
M18 (SEQ ID NO: 37 und 38)	2,9
M19 (SEQ ID NO: 39 und 40)	2,0
M20 (SEQ ID NO: 41 und 42)	2,2
M21 (SEQ ID NO: 43 und 44)	2,3
M22 (SEQ ID NO: 45 und 46)	3,7
M23 (SEQ ID NO: 47 und 48)	5,3
M24 (SEQ ID NO: 1 und 2)	6,2
M25 (SEQ ID NO: 3 und 4)	2,9
M26 (SEQ ID NO: 5 und 6)	3,2
M27 (SEQ ID NO: 7 und 8)	2,0
M28 (SEQ ID NO: 9 und 10)	2,1
M29 (SEQ ID NO: 11 und 12)	2,6
M30 (SEQ ID NO: 13 und 14)	3,4
M31 (SEQ ID NO: 15 und 16)	2,0
M32 (SEQ ID NO: 19 und 20)	3,0
M33 (SEQ ID NO: 17 und 18)	2,0

Tabelle 3

Pharmacological properties of bifunctional urokinase variants M12 and M23 in animal studies

In a pharmacological in vivo model, the effect of bifunctional urokinase variants M12 and M23 on arterial vasoconstriction thrombolysis was tested in comparison to saruplasma (unglycosylated prourokinase) in anesthetized rabbits in a temporarily isolated approximately 1 cm segment of the femoral artery, injected locally via a side thrombin and 125J-labelled human fibrinogen, resulting in the formation of a thrombus leading to complete vascular blockage. The gross amounts of the different thrombus were determined by the combined radioactivity of the gamma-fibrose group via an extracorporeal plasma detector. The measurement of the radioactivity of the gamma-fibrose group was carried out by an extracorporeal plasma detector. The measurement of the radioactivity of the blood of the human fibrinose group was successfully performed by continuous absorption of the blood of the fibrose receptor and the radioactivity of the urine was determined by the addition of 60 mg of M12 and 623 mg of M23-fibrose. The effects were measured in the total time between administration of the serum and the blood of the serum.

During the 90-minute test duration, thrombolysis of the labelled thrombus fibrin was 46 ± 11% for M12, 43 ± 12% for M23, 22 ± 5% for saruplase and 39 ± 15% for the saruplase-heparin combination. Bolus applications of M12 and M23 resulted in the opening of the thrombotically closed vessel in all 6 animals; saruplase application resulted in the vessel reflux in 5 of 6 animals and saruplase and heparin application in 4 of 6 animals. The maximum reperfusion flow rate (in % of baseline) was 95 ± 10% for M12 and 82 ± 93% for M23 and significantly differed from the maximum reperfusion rate of saruplase for itself by 43 ± 12%.The maximum level of reperfusion flow of 58 ± 8% for Saruplase and heparin was between the results of M12 and M23 and Saruplase and did not differ significantly between the two groups. The total fibrinolytic effect was measured as the area of reperfusion flow (as % of the initial flow) over the 90-minute test duration. This total effect was 4.502 ± 1.127 %·min for M12 and 4.270 ± 885 %·min for M23 and was significantly greater for both urokinase variants of the invention than the 1.519 ± 643 %·min for Saruplase.The results of the M12 and M23 studies were summarized in Table 4 and the results of the M12 and M23 studies were not significantly better than those of the Saruplase alone. Tabelle 4

Thrombolytische Wirkung nach i.v.-Bolusapplikation; Femoralarterien-Thrombose, narkotisiertes Kaninchen.
Polypeptid	Dosis		Maximaler Reperfusionsfluß (% des Vorwertes)	Kumulativer Reperfusionsfluß (% · min)
M12	6 mg/kg	46 ± 11	95 ± 10*	4502 ± 1127
M23	6 mg/kg	43 ± 12	82 ± 9*	4270 ± 885*
Saruplase	6 mg/kg	22 ± 5	43 ± 12	1519 ± 643
Saruplase + Heparin	6 mg/kg 150 U/kg	39 ± 15	58 ± 8	2217 ± 761

Tabelle 4

* p <0.05 vs Saruplase

Surprisingly, both after bolus application of M12 and after bolus application of M23 plasma fibrinogen concentrations were found to decrease significantly less than after bolus application of saruplase. The results are summarized in Table 5. Other Tabelle 5

Wirkung der Bolusapplikationen von M12 und M23 im Vergleich zu Saruplase, ohne und mit Heparin, auf die Abnahme der Plasma-Fibrinogenkonzentration; narkotisierte Kaninchen
Polypeptid	Dosis	Abnahme an Plasma-Fibrinogen (%-Änderung gegenüber Ausgangswert)
		Zeit nach Applikation
		30 min	60 min	90 min
M12	6 mg/kg	-19 ± 9	-20 ± 9*	-19 ± 9*
M23	6 mg/kg	-20 ± 11*	-21 ± 11*	-20 ± 11*
Saruplase	6 mg/kg	-64 ± 7	-66 ± 6	-67 ± 6
Saruplase + Heparin	6 mg/kg + 150 U/kg		-46 ± 8	-45 ± 9

Tabelle 5

* p<0.05 vs Saruplase

The results show that the bifunctional urokinase variants M12 and M23 of the invention dissolve arterial thrombuses which cause complete vascular blockage and restore blood flow to the thrombosed vessels. This effect was achieved by single bolus application of M12 or M23 in non-heparinised animals. Surprisingly, this stronger fibrinolytic effect of M12 and M23 compared to saruplase was associated with a lower consumption of plasma fibrinogen. This means that M12 and M23 have a significantly higher fibrin specificity compared to saruplase.

The improved conservation of plasma fibrinogen by M12 and M23 compared to saruplase means that blood clotting is better maintained and thus the risk of uncontrolled bleeding as a possible complication of systemic fibrinogen degradation is reduced.

Claims (17)

1. Bifunctional urokinase variants of the general formula I    M4-X₁-Y₁ in which

   M4   means the amino acid sequence ⁴⁷Ser to ⁴¹¹Leu of the nonglycosylated prourokinase according to Figure 1 (SEQ ID No.: 21 and 22),

   X₁   is a direct bond between M4 and Y₁ or represents

   a peptide having the sequence Ser-Pro-Pro-Ser-Pro-Pro-Gly-Gly-Phe or Ser-Pro-Pro-Ser-Pro-Pro-Ser-Pro-Pro-Gly-Gly-Phe or Ser-Pro-Pro-Ser-Pro-Pro-Ser-Pro-Pro-Gly-Gly-Phe-Gly or

   a peptide sequence of the general formula II    Ser-X₂-X₃-X₄-X₅-X₆-X₇ wherein X₂ means Pro or Leu, X₃ means Val or Pro, X₄ means Lys, Val, Arg, Gly or Glu, X₅ means Ala, Val, Gly, Leu or Lle, X₆ means Phe, Trp, Tyr or Val and X₇ means Gly or a direct bond between X₆ and Y₁

   and

   Y₁   represents a peptide having the sequence Y₂-Arg-Pro-Y₃-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Y₄ or Y₂-Arg-Pro-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-Trp-Glu-Asp-Glu-Glu-Lys-Asn-Glu or Y₂-Arg-Pro-Ser-Ser-Glu-Phe-Glu-Glu-Phe-Glu-Ile-Asp-Glu-Glu-Glu-Lys with Y₂ being Pro or Val, Y₃ Leu or a direct bond between Pro and Gly and Y₄ Gln or a hydroxyl group.
2. Urokinase variants according to claim 1, characterised in that Y₁ means a peptide having the sequence Y₂-Arg-Pro-Y₃-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Y₄.
3. Urokinase variants according to claim 1, characterised in that Y₁ means a peptide having the sequence Y₂-Arg-Pro-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-Trp-Glu-Asp-Glu-Glu-Lys-Asn-Glu.
4. Urokinase variants according to claim 1 and/or claim 2, characterised in that X₁ represents a peptide sequence of the general formula II, in which X₂ means Pro or Leu, X₃ means Val, X₄ means Lys, Val or Arg, X₅ means Ala, Val or Gly, X₆ means Phe, Trp, Tyr or Val and X₇ means Gly or a direct bond between X₆ and Y₁.
5. Urokinase variants according to claim 4, characterised in that X₄ means Lys or Val, X₅ means Ala or Val, X₆ means Phe, Trp or Tyr and X₇ means Gly or a direct bond between X₆ and Y₁.
6. Urokinase variants according to claim 4 and/or claim 5, characterised in that X₇ is a direct bond between X₆ and Y₁.
7. Urokinase variants according to claim 1 and/or claim 3, characterised in that X₁ represents a peptide sequence of the general formula II, in which X₂ means Pro or Leu, X₃ means Val, X₄ means Lys or Val, X₅ means Ala or Val, X₆ means Phe or Trp and X₇ means a direct bond between X₆ and Y₁.
8. Plasmids for use in obtaining a bifunctional urokinase variant according to claims 1 to 7 characterised in that the operon has a controllable promoter, a Shine-Dalgarno sequence effective as a ribosome binding site, a start codon, a synthetic structural gene for a bifunctional urokinase variant of the general formula I according to claims 1 to 7 and, downstream from the structural gene, one or two terminators and that the plasmids are suitable for expressing the bifunctional urokinase variant in strains of Escherichia coli.
9. Plasmids according to claim 8, characterised in that the distance between the Shine-Dalgarno sequence and the start codon is 6-12, preferably 8-10, nucleotides.
10. Plasmids according to claim 8 and/or claim 9, selected from the group pSJ 69, pSJ 76, PSJ 77, pSJ 78, pSJ 79, pSJ 81, PSJ 83, pSJ 90, pSJ 91, pSJ 92, pSJ 93, pSJ 94, pSJ 95, pSJ 101, pSJ 102, pSJ 103, PSJ 104, pSJ 105, pSJ 106, pSJ 109, pSJ 111, pSJ 114 and pSJ 113.
11. Plasmids according to claim 10, selected from the group pSJ 76, pSJ 81, pSJ 83, pSJ 90, pSJ 91, pSJ 92, pSJ 93, pSJ 94, pSJ 95, pSJ 101, pSJ 102, pSJ 103, pSJ 105, pSJ 106, pSJ 109, pSJ 111 and pSJ 114.
12. Plasmids according to claim 10 and/or claim 11, selected from the group pSJ 76, pSJ 81, pSJ 83, pSJ 91, pSJ 92, pSJ 94, pSJ 95, pSJ 101, pSJ 102, pSJ 103, pSJ 106, pSJ 109, pSJ 111 and pSJ 114.
13. Plasmids according to one or more of claims 10 to 12, selected from the group pSJ 76, pSJ 94, pSJ 95, pSJ 101, pSJ 102, pSJ 103, pSJ 106, pSJ 109, pSJ 111 and pSJ 114.
14. Process for the production of plasmids according to claims 8 to 13, characterised in that they are obtained from the plasmids pBlueskript KS II+, pUC 8 and pGR 201 according to Figures 2 and 2a to 2p.
15. Use of a plasmid according to claims 8 to 13 in obtaining a bifunctional urokinase variant of the general formula I according to claims 1 to 7, characterised in that an Escherichia coli strain is transformed with a plasmid in a manner known per se, expression of the structural gene is induced, the resultant precursor protein of the bifunctional urokinase variant of the general formula I is separated from the medium and the lysed bacterial cells, the precursor protein is solubilised and then refolded under the action of a redox system to yield the polypeptide of the general formula I.
16. Thrombolytic which contains a bifunctional urokinase variant of the general formula I according to claims 1 to 7 as the active substance.
17. Thrombolytic according to claim 16, characterised in that it is suitable for bolus administration.