SI22865A - Peptide uroaktivin as activator of enzyme urokinase - Google Patents

Abstract

During intensive studies leading to the submitted invention, the inventors here found out that the peptide uroaktivin with the amino acid sequence SEQ ID NO: 1 binds and increases the enzymatic activity of urokinase and tissue plasminogenic activator. Thus according to the first aspect, the submitted invention assures the amino acid sequence of the peptide uroaktivin - SEQ ID NO: 1. The submitted invention also assures the use of the peptide uroaktivin for a more successful treatment of diseases or states in which the application of thrombolytics is indicated - such states are particularly acute coronary thromboses (acute myocardial infarction), acute ischemic brain strokes, acute pulmonary thromboembolisms, deep venous thromboses and acute arterial thromboembolisms - and for assuring patency of arteriovenous canules and intravenous catheters.

Description

PEPTID UROACTIVIN AS AN ACTIVATOR OF UROKINASE ENCYMS

DESCRIPTION OF THE INVENTION

A. TECHNICAL FIELD OF THE INVENTION

The present invention relates to a peptide uroactivin that binds to a urokinase plasminogen activator (urokinase) and enhances its enzymatic activity. In particular, the present invention relates to the use of such a peptide for the treatment of all diseases using a recombinant tissue or urokinase plasminogen activator, that is, in all indications of the use of thrombolytics. The present invention relates generally to thrombolytics, in particular tissue and urokinase plasminogen activators.

B. DISPLAY OF THE PROBLEMS SOLVED BY THE INVENTION

Unwanted vascular clot formation (thrombosis) is one of the major cardiovascular complications. The formation of a clot (thrombus) can lead to a decrease or complete interruption (occlusion) of blood flow through the vessel. Interruption of blood flow can lead to acute myocardial infarction, ischemic stroke, venous thromboembolism or pulmonary embolism. Anti-thrombotic agents are used to prevent clot formation, including anticoagulants (vitamin K antagonists), platelet aggregation inhibitors (acetylsalicylic acid, heparin and heparin derivatives, hirudin, clopidogrel, dipyridamole) and thrombolytics (or fibrinolytics). Clots in veins where blood flow is slower are composed mainly of cross-linked fibrin and less of platelets, so the primary therapy is warfarin and heparin type anticoagulants. Clots in the arteries where blood flow is faster contain more platelets and less fibrin, so acetylsalicylic acid is used as a preventative, and thrombolytic agents are used in the case of occlusion. Anticoagulants and platelet aggregation inhibitors prevent new clots from forming and enlarging pre-existing clots, while thrombolytics are used to break down (lick) obstructive clots as quickly as possible, without preventing any new clots from forming.

Physiological fibrinolysis

Fibrinolysis is a normal process in the body that goes hand in hand with coagulation, allowing for haemostasis and preventing blood clots that would cause blood flow restrictions. In the process of coagulation from fibrinogen under the influence of thrombin, fibrin monomers are formed, which polymerize and form protofibrils constructed from two antiparallel non-covalently linked chains. Within one chain, the fibrin monomers are covalently linked. The major fibrinolytic enzyme is the plasmin serine protease, which is generated by plasminogen through activators. Plasminogen contains secondary structure motifs that specifically bind to lysine and arginine fibrin (ogen) a and are incorporated into fibrin chains during clot formation. In the process of fibrinolysis, plasminogen is converted to the active form and cleaves fibrin C-terminally with respect to bound lysines and arginines. Initially, plasmin creates gaps in fibrin, and further degradation causes solubilization of fibrin. (Walker and Nesheim, 1999).

The primary plasminogen activators are tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA, urokinase). tPA is released from vascular endothelial cells and urokinase is released from fibroblasts, endothelial cells and monocytes. The process of fibrinolysis regulates a large number of inhibitors (plasminogen activator inhibitor-lin -2, PAI-1, PAI-2; alpha2-antiplasmin; alpha2-macroglobulin; serpin; TAFI), ensuring that lysis is locally restricted.

Thrombolytic agents and their indications

Fibronilzo can be triggered pharmacologically by e.g. thrombolytic (or fibrinolytic) therapy. The most commonly used thrombolytics for systemic use are:

- alteplase (recombinant tPA)

- aniztreplaza

- streptokinase and

- urokinase.

In addition, some thrombolytic agents are used elsewhere:

- staphylokinase (bacterial recombinant protein)

- duteplazase (tPa composed of two protein chains)

reteplase (a tPA - like protein with a longer elimination half - life), and

- tenecteplase (tPA with two changed amino acids).

Due to the protein nature, thrombolytic agents are only administered parenterally. What is most important in the choice of thrombolytic therapy is that it should be based on the evaluation of the individual's thrombotic complication, the individual's condition and his / her history. When using thrombolytics, a balance between the potential benefit and the risk of haemorrhage, a major adverse effect of thrombolysis, must be considered.

Thrombolytics are used to treat:

- acute coronary thrombosis (acute myocardial infarction)

- acute ischemic strokes

- acute pulmonary thromboembolism

- deep vein thrombosis

- acute arterial thromboembolism

- acute arterial thrombosis.

Thrombolytics are used to ensure transience:

- arteriovenous cannulas

- intravenous catheters.

Urokinase and its action

Urokinase is a 411 amino acid large protein composed of three domains: a C-terminal catalytic domain containing the active site of a serine protease, an N-terminal kringle domain, and a domain homologous to the epidermal growth factor that binds to the UPAR / CD87 receptor.

Urokinase is synthesized in the form of zymogen (prourokinase, pro-uPA and single-chain urokinase, sc-uPA) and is activated by proteolytic cleavage between Lys [158] -Ile [l59]. The resulting chains remain connected via a disulfide bond.

Urokinase directly cleaves the peptide bond Arg [560] -Val [561] in plasminogen, producing plasmin and activating the fibrinolytic system. Plasmin breaks down fibrin, fibrinogen and other plasma proteins in clots, including coagulation factors V and VIII.

Single- and double-stranded forms of urokinase bind to plasma membrane receptors: uPAR / CD87 and LDL receptor family receptors: LDLR-relative protein, alpha2macroglobulin receptor (LRP / a ₂ -MR), and VLDL receptor. The binding affinity of urokinase to the LDL receptor family (Kd ~ (1-2) -10 ^-8 M) receptors is a size class lower than binding to uPAR / CD87. In addition, urokinase can bind to some other receptors via the protease and kringle domains.

The multidomain structure facilitates the polyfunctional action of urokinase, as it contains numerous binding sites for both substrates and different cellular receptors.

Tissue plasminogen activator and its action

Tissue plasminogen activator (tPA) is a serine protease and is classified as urokinase in the Sl A family of serine proteases. It is normally found on the surface of endothelial cells of the veins, capillaries, pulmonary arteries, heart and uterus and is secreted after damage to the vein.

tPA is synthesized as a single-stranded polypeptide and cleaves after disintegration of plasmin or trypsin (cleavage of the Arg-Ile bond) into a double-stranded polypeptide linked to a disulfide bond. The tPA heavy chain is located at the N-terminal end (Mr 39,000) and the light chain is at the C-terminal end (Mr 33,000). tPA consists of five different structural domains: a domain homologous to a fibronectin fmger-like structure, a domain homologous to the epidermal growth factor, kringel domains 1 and 2 (as found in urokinase, plasminogen, thrombin, factor XII) are in the heavy chain and the active site a light chain serine protease.

A tissue plasminogen activator converts plasminogen into plasmin. The specificity of ativation depends on the affinity of tPA to fibrin and the formation of a ternary complex between tPA, plasminogen, and fibrin, resulting in a strong increase in the plasminogen activation rate on the fibrin surface. The functional tPA domains responsible for affinity for fibrin and enhancement of tPA activity are located within the N-terminal region (heavy chain), most likely in the fingerlike domain (Binyai) and in the second kringel domain (van Zooneveld).

Role of cytokeratins in plasminogen activation

Activation of plasminogen on the cell surface is much faster than activation in solution, since the interaction of plasminogen with binding proteins results in a change in the conformation of plasminogen, which is more open to proteolysis (Ellis, 1991). Cytokeratin 8 is a major plasminogen binding protein on the membrane of breast cancer cells. Cytokeratin 8 is an intermediate filamentous protein that, in combination with cytokeratin 18, forms an insoluble structural cytoplasmic skeleton. The C-terminal portion of cytokeratin 8 penetrates the cell membrane (Hembrough, 1995; Ditzel, 1997) and binds plasminogen (Kralovich, 1998). Plasminogen activation is accelerated by the binding of tissue plasminogen activator (tPA), which can bind to either cytokeratin 8 or cytokeratin 18 (Kralovich, 1998)

The peptide sequence of uroactivin corresponds to the amino acid sequence located in the Cterminal portion of cytokeratins 1, 2, 8, 10 and 18 as a strongly conserved region of cytokeratins. This region is assumed to be the binding site for tPA as well as urokinase.

C. DETAILS OF TECHNICAL STATUS

The state of the art in the field of the use of urokinase for the breakdown of unwanted clots in the body (fibrinolysis) is described by the following sources, but according to the data available to date, urokinase activators have not yet been patented:

- Banyai L, Varadi A, Patthy L. Common evolutionary origins of the fibrin-binding structures of a fibronectin and tissue-type plasminogen activator. FEBS Lett. 1983 Oct 31; 163 (1): 37-41.

- Ditzel H, Garrigues U, Andersen C, Larsen M, Garrigues H, Svejgaard A, Hellstrom I, Hellstrom K, Jensenius J: Modified cytokeratins expressed on the surface of carcinoma whole undergo endocytosis upon binding of the human monoclonal antibody and its recombinant Fab fragment . Away Natl Acad Sci U S A 1997, 94 (15): 8110-8115.

- Ellis V, Behrendt N, Dano K: Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor. J Biol Chem 1991, 266 (19): 12752-12758.

- Hembrough T, Vasudevan J, Allietta M, Glass Wn, Gonias S: A cytokeratin 8-like protein with plasminogen-binding activity is present on the extemal surfaces of hepatocytes, HepG2 whole and breast carcinoma whole lines. J Celi Sci 1995, 108 (Pt 3): 1071–1082.

- Kralovich KR, Li L, Hembrough TA, Webb DJ, Kams LR, Gonias SL. Characterization of binding sites for plasminogen and tissue-type plasminogen activator in cytokeratin 8 and cytokeratin 18. J Protein Chem. 1998 Nov; 17 (8): 845-54.

- Van Zonneveld A J, Veerman H, Pannekoek H. Autonomous functions of structural domains on human tissue-type plasminogen activator. Away from Natl Acad Sci USA. 1986 Jul; 83 (13): 4670-4.

Walker JB, Nesheim ME. Molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. J Biol Chem. 1999 Feb 19; 274 (8): 5201 -12.

United States Patent 6,861,054; Higazi. March 1, 2005, suPAR stimulating activity of tcuPA-mediated fibrinolysis and different uses.

United States Patent 4,996,050; Tsukada, et al. February 26, 1991: Fibrinolytic activity enhancer.

D. DESCRIPTION OF THE DRAWINGS OF THE INVENTION

Figure 1 shows the results of binding of the uroactivin peptide to the urokinase plasminogen activator obtained by surface plasmon resonance. The curves represent binding of the urokinase plasminogen activator to immobilized uroactivin and its dissociation at concentrations of 10 nM, 20 nM and 50 nM (bottom up).

Figure 2 shows plasminogen activation in the presence of the peptide uroactivin. Plasminogen activation was followed by measuring fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, plasmid specific substrate D-Ala-Lev-Lys-AMC (0.5 mM), and increasing peptide concentrations. Fluorescence was read after 90 min.

Figure 3 shows the kinetics of plasminogen activation in the presence of the peptide uroactin. Plasminogen activation was followed by measuring fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, plasmid specific substrate D-Ala-Lev-Lys-AMC (0.5 mM), and increasing peptide concentrations. Fluorescence was monitored for 100 min.

Figure 4 shows plasminogen activation in the presence of the peptide uroactivin. Plasminogen activation was followed by measuring fluorescence at 470 nm in the presence of 500 pM tissue plasminogen activator, 500 nM plasminogen, specific plasmid substrate D-Ala-Lev-Lys-AMC (0.5 mM), and increasing peptide concentrations. Fluorescence was read after 90 min.

Figure 5 shows the kinetics of plasminogen activation in the presence of the peptide uroactin. Plasminogen activation was followed by measuring fluorescence at 470 nm in the presence of 500 pM tissue plasminogen activator, 500 nM plasminogen, specific plasmid substrate D-Ala-Lev-Lys-AMC (0.5 mM), and increasing peptide concentrations. Fluorescence was monitored for 100 min.

Figure 6 shows a control experiment of plasminogen activation in the presence of a peptide of uroactivin and in the absence of a plasminogen activator (uPA or tPA). Plasminogen activation was followed by measuring fluorescence at 470 nm in the presence of 500 nM plasminogen, a specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM), and increasing peptide concentrations. Fluorescence was read after 90 min.

Figure 7 shows a control experiment of plasminogen activation in the presence of undecapeptide. Plasminogen activation was followed by fluorescence measurements at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, plasmid specific substrate D-Ala-Lev-Lys-AMC (0.5 mM), and increasing undecapeptide concentrations. Fluorescence was read after 90 min.

Figure 8 shows an attempt to break down a fibrin clot in the presence of a peptide of uroactivin. The degradation of the fibrin clot was followed by measuring the absorbance at 405 nm in the presence of 500 nM plasminogen, a urokinase plasminogen activator (250 pM, 500 pM and 1 nM) and increasing concentrations of uroactin. Absorbance was monitored until the fibrin clot was completely exposed.

E. IMPLEMENTING EXAMPLES

The present invention is illustrated by the following non-limiting examples:

1. Design of peptides

The amino acid sequence of cytokeratins 1, 2, 8, 10 and 18 is derived from the NCBI Protein Database protein database (1346343, 547754, 225702, 40354192, and 30311). The sequences were edited using the ClustalW multiple sequence alignment software (www.ch.embnet.org), and based on the ordered sequences, a sequence of the peptide uroactivin that did not correspond to any of the cytokeratin sequences was designed. Also, the uroactin sequence is not in the Blast database of the known amino acid sequences of any living thing so far.

2. Peptide synthesis

The peptide uroactin with the amino acid sequence of SEQ ID NO: 1 was synthesized by Biosynthesis Inc. (Denver, TX, USA). Its purity was 85.6% determined by HPLC and mass analysis.

3. Testing the binding of uroactin to plasminogen activator by surface plasmon resonance

Urokinase binding to uroactivin was determined using a Biacore X system (Biacore, Sweden). The experiment was run on a CM5 sensor chip (BR-1003-98 BIAcore). Uroactivin was immobilized at a flow rate of 1 pL / min for 10 minutes (300 RU). The flow cell was then washed with 5 µL of 10 mM glycine buffer (pH 2.2) at a flow rate of 30 µL / min. The second flow cell was used as the reference cell. The same rinsing process was used to regenerate the dodecapeptide surface during single injections of different peptide concentrations. uPA was dissolved in PBST buffer (PBS with 0.05% Tween 20).

Bound uroactin was treated with different concentrations of plasminogen activator. In the case of the urokinase plasminogen activator, 10 nM, 20 nM, and 50 nM were used.

All measurements were performed at 25 ° C and a flow rate of 1 pL / min in 1% PBST buffer. 5 pL uPA was injected in a single measurement. The association and dissociation curves of the urokinase plasminogen activator on the peptide uroactivin are shown in Figure 1. Using the moving baseline method - the Biacore software package, we determined Ka = 9.8 nM.

4. Plasminogen activation

Accelerated plasminogen activation in the presence of the peptide uroactivin was followed by fluorescence measurements at 470 nm and coincided with increased plasmin production. 500 pM urokinase or tissue plasminogen activator, 500 nM plasminogen, and 0.5 mM specific plasmid substrate D-Ala-Lev-Lys-AMC were used in the assay. The amount of plasmin produced in 90 min and the kinetics of its formation indicate a markedly increased formation in the presence of the peptide uroactivin. Plasmin formation is dependent on the concentration of the peptide uroactivin (Figures 2-5). In control experiments where plasminogen activator was absent, plasmin production did not occur.

uPA

Urokinase plasminogen activator (uPA) (500pM) was dissolved in phosphate buffer, pH 7.4, and 50 µl / well was added. Different concentrations of uroactivin (0.1 μΜ, 0.25 μΜ, 0.5 μΜ, 1 μΜ, 2.5 μΜ, and 5 μΜ) were added and incubated at 37 ° C for 30 min. Subsequently, a specific fluorogenic substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and finally plasminogen (500 nM) (all from Sigma, St. Louis, MD) were added to individual well microtiter plates and followed by plasmin formation for 1.5 hours at 37 ° C (Ex = 370 nm and Em = 470 nm) using a fluorescence microtiter plate reader (Safire 2, Tecan). In the control experiment, undecapeptide was added. dodecapeptide with a different sequence. We also performed a control experiment where we did not add uPA. The result is representative of three independent experiments. The mean values of the four parallels ± standard deviation are given.

tPA

Tissue plasminogen activator (tPA) (500pM) was dissolved in phosphate buffer pH 7.4 and 50 μΐ / well was added. Different concentrations of uroactivin (0.1 μΜ, 0.25 μΜ, 0.5 μΜ, 1 μΜ, 2.5 μΜ, and 5 μΜ) were added and incubated at 37 ° C for 30 min. Subsequently, a specific fluorogenic substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and finally plasminogen (500 nM) (all from Sigma, St. Louis, MD) were added to individual well microtiter plates and followed by plasmin formation for 1.5 hours at 37 ° C (Ex = 370 nm and Em = 470 nm) using a fluorescence microtiter plate reader (Safire 2, Tecan). In the control experiment, undecapeptide was added. dodecapeptide with a different sequence. We also performed a control experiment where no tPA was added. The result is representative of three independent experiments. The mean values of the four parallels ± standard deviation are given.

5. Fibrin clot dissolution test

Fibrin clot was made by adding 50 μΐ human thrombin (0.3 U / ml, final concentration) (1NI U = 0.324 ± 0.073 pg thrombin) and CaCl ₂ (20 mM) to 50 μΐ human fibrinogen (2 mg / ml, final concentration) in 96-well plate. The clot formation took place at room temperature for 3 hours. Then, 50 μΐ 1 μΜ plasminogen and 50 μ 50 urokinase plasminogen activator (500 pM, 1.0 nM, 2.0 nM) were added with the addition of uroactin (20 μΜ, 10 μΜ, 5 μΜ, 0 M). Changes in clot opacity were monitored by measuring the change in absorbance over time at 405 nm at 25 ° C on a microtiter reader. The measurements were performed in four parallel units for each concentration and repeated three times.

Ί2>

SEQUENCE LISTING <110> OBERMAJER Natasa, DOLJAK Bojan, KOS Janko, UNIVERSITY OF LJUBLJANA <120> Peptide uroactivin, as an activator of enzyme urokinase (Peptide uroactivin, as an activator of the urokinase enzyme) <140> P-200800224 <141> 2008-09- 2008 25 <160> 1 <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220>

<223> Designed peptide based on a highly homologous region of OKI, CK2, CK8, CK10 and CK18.

<400> 1

Val Lys Ile Ala Leu Glu Val Lys Ile Ala Thr Tyr

Claims (11)

An amino acid sequence in uroactivin (a peptide having the amino acid sequence of SEQ ID NO. 1) and peptidomimetics derived from the sequence. 2. A pharmaceutical composition comprising uroactivin or peptidomimetics derived therefrom. A pharmaceutical formulation comprising uroactivin and peptidomimetics derived therefrom according to any one of claims 1-2, which permits parenteral administration. A medical device comprising uroactin and peptidomimetics derived therefrom according to any one of claims 1-2, for use in the direct or indirect prevention of blood clotting, especially in catheters and vascular implants. The peptide uroactin with the amino acid sequence of SEQ ID NO.l or peptidomimetics derived therefrom according to claim 1 for use as an activator of enzymes that accelerate the fibrinolysis process. A peptide uroactin with the amino acid sequence of SEQ ID NO.l or peptidomimetics derived therefrom according to claim 1 for use as a urokinase plasminogen activator. 7. The peptide of uroactin with the amino acid sequence of SEQ ID NO. 1 or peptidomimetics derived therefrom according to claim 1 for use as a tissue plasminogen activator. 8. The peptide uroactin with the amino acid sequence of SEQ ID NO.l or peptidomimetics derived therefrom according to claim 1 for use as a medicament. A pharmaceutical composition comprising the peptide uroactin with the amino acid sequence of SEQ ID NO. 1 or peptidomimetics derived therefrom according to claim 1 for use as an activator of enzymes that accelerate the fibrinolysis process, as an activator of a urokinase plasminogen activator or as a tissue plasminogen activator. Use of the peptide uroactin with the amino acid sequence of SEQ ID NO. 1 or peptidomimetics derived therefrom according to claim 1, for the manufacture of a pharmaceutical composition for the treatment of diseases or conditions that indicate the use of platelet. Use of a uroactin peptide with the amino acid sequence of SEQ ID NO. 1 or peptidomimetics derived therefrom according to claim 1, for the manufacture of a pharmaceutical composition for the treatment of acute coronary thrombosis (acute myocardial infarction), acute ischemic stroke, acute pulmonary thromboembolism, deep vein thrombosis, acute arterial thromboembolism, and acute thromboembolism, patency of arteriovenous cannulas and intravenous catheters.