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WO2010034490A1 - Peptide uroaktivin, as an activator of the enzyme urokinase - Google Patents

Peptide uroaktivin, as an activator of the enzyme urokinase Download PDF

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Publication number
WO2010034490A1
WO2010034490A1 PCT/EP2009/006899 EP2009006899W WO2010034490A1 WO 2010034490 A1 WO2010034490 A1 WO 2010034490A1 EP 2009006899 W EP2009006899 W EP 2009006899W WO 2010034490 A1 WO2010034490 A1 WO 2010034490A1
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Prior art keywords
uroaktivin
amino acid
acid sequence
peptide
peptidomimetics
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Natasa Obermajer
Bojan Doljak
Janko Kos
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Univerza Ljubljana v Fakulteta za Farmazijo
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Univerza Ljubljana v Fakulteta za Farmazijo
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the peptide urogen, that binds to the urokinase type plasminogen activator (urokinase) and as a consequence of binding increases the enzymatic activity of urokinase.
  • the present invention relates to the use of said peptide for the therapy of conditions and diseases, that are usually treated with recombinant tissue type or urokinase type plasminogen activator, i.e. all the indications that require thrombolytic medicines.
  • the present invention relates in general to thrombolytic medicines, especially to urokinase type plasminogen activator.
  • thrombosis blood vessels
  • thrombus a coagulation clot
  • the interruption of blood flow may lead to an acute myocardial infarction, ischemic brain infarction, venous thromboembolism or lung embolism.
  • Antithrombotic medicines are used for the prevention of clot formation, among which there are anticoagulants (vitamin K antagonist), inhibitors of thrombocyte aggregation (acetylsalicylic acid, heparin and heparin derivatives, hirudin, clopidogrel, dipiridamol) and thrombolytics (fibrinolytics).
  • anticoagulants vitamin K antagonist
  • inhibitors of thrombocyte aggregation acetylsalicylic acid, heparin and heparin derivatives, hirudin, clopidogrel, dipiridamol
  • thrombolytics fibrinolytics
  • acetylsalicylic acid is used for prophylaxis of arterial thrombi or in case of occlusion - thrombolytic medicines.
  • Anticoagulants and inhibitors of thrombocyte coagulation prevent the formation of new and the enlargement of present thrombi, while thrombolytic medicines are adopted for the immediate break-up (lysis) of obstructive clots, but they do not prevent the formation of new thrombi.
  • Fibrinolysis is a normal process in the body, that is going on simultaneously with coagulation and preserves homeostasis by preventing the formation of thrombi, that would obstruct the flow of blood.
  • fibrinogen is transformed by thrombin to fibrin monomers, that polymerize and form protofibirls, composed of two antiparallel non- covalently linked chains. Inside each chain, the fibrin monomers are linked together covalently.
  • the main fibrinolytic enzyme is the serine protease plasmin, which is generated from plasminogen by activators.
  • Plasminogen contains secondary structure motifs that specifically bind lysine and arginine of fibrin(ogen) which enables plasminogen to bind to the network of fibrin during thrombus formation.
  • plasminogen is transformed into its active form and cleaves fibrin on the C-terminal end in regard to the bound lysines and arginines.
  • plasmin initially creates gaps in the fibrin network with further degradation resulting in the complete solubilisation of fibrin (Walker, 1999).
  • plasminogen tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA, urokinase).
  • tPA tissue type plasminogen activator
  • uPA urokinase type plasminogen activator
  • tPA is released from endothelial cells of blood vessels, while urokinase is released from fibroblasts, endothelial cells and monocytes.
  • the process of fibrinolysis is regulated by many inhibitors (inhibitor of plasminogen activator-1 and -2, PAI-I, PAI-2; alpha2-antiplasmin; alfa2-macroglobulin; serpin; TAFI), which enable the localisation of the lysis.
  • inhibitors inhibitor of plasminogen activator-1 and -2, PAI-I, PAI-2; alpha2-antiplasmin; alfa2-macroglobulin; serpin; TAFI
  • Fibrinolysis can be triggered pharmacologically with the so called thrombolytic (fibrinolytic) therapy.
  • thrombolytic fibrinolytic
  • the most commonly used thrombolytics for systemic use are:
  • tPA duteplase
  • reteplase a tPA-similar protein with a longer half time of elimination
  • thrombolytic medicines are applied parenterally, due to their protein nature.
  • the most important aspect of thrombolytic therapy is that it must be based on the individual characterisation of the thrombotic complication, the individual's status and his/her anamnesis.
  • thrombolytics it must be taken into consideration the balance between their potential positive effects and the risk of bleeding, which is the main adverse effect of thrombolysis.
  • Thrombolytics are used for the treatment of:
  • Thrombolytics are used for ensuring the permeability of:
  • Urokinase is a 411 amino acid long protein composed of three domains: C-terminal catalytical domain with the active serine protease site, N-terminal Kringle domain and the epidermal growth factor domain, that binds to the uPAR/CD87 receptor.
  • Urokinase is synthesized in the form of a zymogen (prourokinase - pro-uPA and single-chain urokinase - sc-uPA) and is activated by proteolytic cleavage of the Lys[158]-Ile[159] bond. The two resulting chains remain connected via a disulphide bond.
  • Urokinase directly cleaves the peptide bond between amino acids Arg[560]-Val[561] of plasminogen to produce plasmin that in turn activates the fibrinolytic system. Plasmin cleaves fibrin, fibrinogen in other plasma proteins in coagulation clots, among others the coagulation factors V and VIII. Single- and double-chain forms of urokinase bind to receptors on the plasma membrane: uPAR/CD87 and receptors of the LDL receptor family: LDLR-relative protein, receptor alfa2-macroglobuline (LRP/ ⁇ 2 -MR) and VLDL- receptor.
  • the affinity of the urokinase binding to the receptors of the LDL receptor family (IQ- (l -2)-10 ⁇ 8 M) is about one degree of magnitude lower than the binding affinity to uPAR/CD87.
  • urokinase can bind to other receptors through its protease and Kringle domain.
  • the multidomain structure of urokinase enables its polyfunctional activity, as it contains many binding sites for substrates and other cell receptors.
  • Tissue type plasminogen activator is a serine protease and like urokinase, a member of the SlA serine protease family. It is normally present on the surface of endothelial cells of veins, capillaries, lung arteries, heart and uterus and is released when the vessel is damaged.
  • tPA is synthesized as a single-chain polypeptide which is converted into a double-chain polypeptide connected with a disulphide bond, after the action of plasmin or trypsin (cleavage of the Arg-Ile bond).
  • the heavy chain of tPA represents the N-terminal part (Mr 39.000), while the light chain represents the C-terminal end (Mr 33.000).
  • tPA is composed of five distinct structural domains: the domain homologous to the fibronectin finger-like structure, domain homologous to the epidermal growth factor, active serine protease site and Kringle domains 1 and 2 (as they are present in urokinase, plasminogen, thrombin and factor XII). All but the active site are situated on the heavy chain of tPA.
  • tPA converts plasminogen into plasmin. The specificity of the activation depends on the affinity of tPA towards fibrin and the formation of a tertiary complex between tPA, plasminogen and fibrin, which results in the strong increase of the activational rate of plasminogen on the fibrin surface.
  • tPA The functional domains of tPA, that are responsible for the affinity towards fibrin and the increase of tPA activity are situated inside the N-terminal region (heavy chain), probably in the finger-like domain (Banyai, 1983) and the Kringle domain 2 (van Zonneveld, 1986).
  • Activation of plasminogen on the cell surface is much faster compared to the activation in solution, as the interaction of plasminogen with binding proteins results in a conformational change of plasminogen to a conformation that is more open for proteolytic action (Ellis, 1991).
  • Cytokeratin 8 is the main binding protein of plasminogen on the membrane of breast cancer cells. Cytokeratin 8 is an intermediate filament protein, which pairs with cytokeratin 18 and forms the insoluble structural cytoplasmic skeleton. The C-terminal part of cytokeratin 8 penetrates through the cell membrane (Hembrough, 1995; Ditzel, 1997) and binds plasminogen (Kralovich, 1998). The activation of plasminogen is accelerated by the binding of tPA to cytokeratin 8 or to cytokeratin 18 (Kralovich, 1998).
  • sequence of peptide uro15in is derived from the highly homologous (although not identical) sequence of the C-terminal part of cytokeratins 1, 2, 8, 10 and 18. We suggest that this region is responsible for the binding of tPA and urokinase.
  • Fig. 1 shows the results of the binding of urooxidin to the urokinase type plasminogen activator acquired by the surface plasmon resonance technique.
  • the curves represent the binding of urokinase type plasminogen activator to the immobilised uroeptin and its dissociation at concentrations 10 nM, 20 nM and 50 nM (in this order from bottom up).
  • Fig. 2 shows the activation of plasminogen in the presence of peptide urooxidin.
  • the activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was measured after a period of 90 min.
  • Fig. 3 shows the kinetics of the activation of plasminogen in the presence of peptide urogenin.
  • the activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was followed for 100 min.
  • Fig. 4 shows the activation of plasminogen in the presence of peptide uroeptin.
  • the activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM tissue type plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was measured after a period of 90 min.
  • Fig. 5 shows the kinetics of the activation of plasminogen in the presence of peptide urogenin.
  • the activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM tissue type plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was followed for 100 min.
  • Fig. 6 shows the control experiment of the activation of plasminogen in the presence of peptide urogen and in the absence of plasminogen activator (uPA or tPA).
  • the activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was measured after a period of 90 min.
  • Fig. 7 shows the control experiment of the activation of plasminogen in the presence of a negative control undekapeptide.
  • the activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of undekapeptide. The fluorescence was measured after a period of 90 min.
  • Fig. 8 shows the experiment of lysis of fibrin clot in the presence of peptide urogenin.
  • the lysis of fibrin clot was determined by measuring the absorbance at 405 nm in the presence of 500 nM plasminogen, urokinase plasminogen activator (250 pM, 500 pM and 1 nM) and increasing concentrations of urogenin. Absorbance was measured until the total lysis of the fibrin clot.
  • the present invention relates to the amino acid sequence in uroeptin (peptide with amino acid sequence SEQ ID NO:1) and peptidomimetics, deduced from the amino acid sequence of uroeptin.
  • Peptidomimetics within the context of this application can be understood as being peptides which are 50, 70, 80, 90, 95, 98, 99, or 99.9% identical to SEQ ID NO:1.
  • Peptidomimetics preferably have the same biochemical function as has SEQ ID NO: 1.
  • the invention thus also relates to peptides with are 50, 70, 80, 90, 95, 98, 99, or 99.9% identical to SEQ ID NO:1.
  • These peptides preferably have the same biochemical function as has SEQ ID NO:1.
  • Peptides of the invention are preferably 8, 9, 10, 11, 12, 13, 15, 20, 30, 50, 100 amino acids in length.
  • % identity of a protein relative to a reference protein of a defined length shall be understood as follows: A peptide that is 50 percent identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide, which is 50 percent identical to the reference polypeptide over its entire length. Of course, other polypeptides will meet the same criteria.
  • sequence identity as used herein means that the sequences are compared as follows.
  • sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default (BLOSUM62) matrix (values -4 to +11) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (per each additional consecutive null in the gap).
  • percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference polypeptide.
  • the invention also relates to DNA coding for the peptides of the invention.
  • the present invention further relates to pharmaceutical composition
  • pharmaceutical composition comprising uroeptin or peptidomimetics, deduced from the amino acid sequence of uroeptin.
  • the pharmaceutical compositions contain uroeptin or peptidomimetics, deduced from the amino acid sequence of uroeptin.
  • the pharmaceutical composition are suitable for parenteral application.
  • the invention further relates to medical equipment, designated to contain urooxidin or peptidomimetics of the invention, which can be used in direct or indirect prevention of blood clotting, in particular in application of catheters and stents.
  • the invention further relates to the use of peptide urodonin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of urodonin, as activator of enzymes, which accelerate the process of fibrinolysis.
  • the invention further relates to the use of eptide urogenin with amino acid sequence SEQ ID NO.l and peptidomimetics deduced from the amino acid sequence of uroeptin as an activator of urokinase type plasminogen activator.
  • the invention further relates to the use of peptide uroeptin with amino acid sequence SEQ ID NO.l and peptidomimetics deduced from the amino acid sequence of uroeptin as activator of tissue type plasminogen activator.
  • the invention further relates to peptide uroeptin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroeptin to be used as a drug.
  • the invention further relates to the use of said peptides as a drug.
  • the invention further relates to a pharmaceutical composition, characterised in that it contains peptide urogen with amino acid sequence SEQ ID NO.1 and/or peptidomimetics, deduced from the amino acid sequence of uroeptin, to be used as activator of enzymes, which accelerate the process of fibrinolysis, as activator of urokinase type plasminogen activator and/or as activator of tissue type plasminogen activator.
  • the invention further relates to the use of peptide uroeptin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroeptin for preparation of pharmaceutical compositions for treatment of diseases and conditions wherein thrombolytics are indicated.
  • the application further relates to the use of peptide urogenin with amino acid sequence SEQ ID N0.1 and peptidomimetics, deduced from the amino acid sequence of urogenin, for preparation of pharmaceutical compositions for treatment of one of acute coronary thrombosis (acute myocardial infarction), acute ischemic brain infarction, deep venous thrombosis, acute lung thromboembolism, acute arterial thromboembolism, acute arterial thrombosis, and for assurance of permeability of arteriovenous cannulas and intravenous catheters.
  • acute coronary thrombosis acute myocardial infarction
  • acute ischemic brain infarction deep venous thrombosis
  • acute lung thromboembolism acute arterial thromboembolism
  • acute arterial thrombosis acute arterial thrombosis
  • the amino acid sequences of cytokeratins 1, 2, 8, 10 in 18 were derived from the NCBI Protein Database (entry # 1346343, 547754, 225702, 40354192 and 30311, respectively). The sequences were aligned with the ClustalW multiple sequence alignment software (www.ch.embnet.org) and on the basis of the aligned sequences, the amino acid sequence of the peptide uro15in was designed. The resulting amino acid sequence does not belong to neither cytokeratins, and does not belong to any of the known living organisms according to the Blast data base (http://blast.ncbi.nlm.mh.gov/Blast.cgi)
  • the peptide uroeptin with the amino acid sequence SEQ ID NO: 1 was synthesized at Biosynthesis Inc. (Denver, TX, USA). The purity of the synthesized peptide was 85.6 % determined by HPLC and mass analysis.
  • Example 3 Testing or the binding of urokinase type plasminogen activator to urogenin with the method of surface plasmon resonance
  • the binding of urokinase to urogenin was determined with a Biacore X system (Biacore, Sweden). The experiment was carried out on a CM5 sensor chip (cat. # BR-1003-98 Biacore). Urozin was immobilised at a constant flow of 1 ⁇ L/min for 10 minutes on the flow cell #2 (up to 300 RU). The flow cell was then washed with 5 ⁇ L 10 mM glycine buffer (pH 2.2) at a flow of 30 ⁇ L/min. The flow cell # 1 was used as a reference cell.
  • uPA was dissolved in a PBST buffer (PBS, pH 7.4 with 0.05% Tween 20).
  • the immobilised urodonin was treated with different concentrations of urokinase: 10 nM, 20 nM and 50 nM. All the measurements were done at 25 0 C and at a flow of 1 ⁇ L/min in 1% PBST buffer. 5 ⁇ L of uPA was injected at each measurement.
  • the association and dissociation curves of urokinase type plasminogen activator on/off the peptide uro15 are shown on Fig. 1. With the drifting baseline method of the Biacore evaluation software, we determined a Ka of 9.8 nM .
  • the increased activation of plasminogen in the presence of peptide urogenin was determined by measuring fluorescence at 470 nm, and matches the increased plasmin formation.
  • Final concentrations of 500 pM urokinase type or tissue type plasminogen activator, 500 nM plasminogen and 0.5 mM specific substrate for plasmin D-Ala-Lev-Lys-AMC were used in the test.
  • the amount of generated plasmin in 90 min and the kinetics of its generation show a strongly increased generation in the presence of peptide uroeptin.
  • the formation of plasmin depends on the concentration of peptide uroeptin (Fig. 2-5). Plasmin was not generated in control experiments, where plasminogen activator was not present.
  • Urokinase type plasminogen activator (50OpM) was dissolved in phosphate buffer, pH 7.4 and added to a 50 ⁇ L well. Urozinin was added in concentrations of 0.1 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2.5 ⁇ M and 5 ⁇ M and incubated at 37 0 C for 30 min. A specific fluorogenic substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and finally plasminogen (500 nM) (all from Sigma, St.
  • Tissue type plasminogen activator 50OpM
  • Urodonin was added in concentrations of 0.1 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2.5 ⁇ M and 5 ⁇ M and incubated at 37 0 C for 30 min.
  • a specific fluorogenic substrate for plasmin D-Ala-Lev-Lys-AMC 0.5 mM
  • plasminogen 500 nM
  • the formation of the clot was carried on for 3 hours at room temperature.
  • 50 ⁇ L of 1 ⁇ M plasminogen and 50 ⁇ L of urokinase plasminogen activator (500 pM, 1.0 nM, 2.0 nM) with uroentin (20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 0 M) were than added. Changes in the opacity of the clot were followed by measuring the absorbance at 405 nm at 25 0 C with a microtiter plate reader. The measurements were done in four parallels and repeated three times.

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Abstract

In the course of intense studies that brought to the present invention, the inventors discovered, that the peptide uroaktivin with the amino acid sequence SEQ ID NO: 1 binds to and increases the activity of urokinase type plasminogen activator (urokinase, uPA) and tissue type plasminogen activator (tPA). The present invention in the first place provides the amino acid sequence of the peptide uroaktivin - SEQ ID NO: 1. The present invention also provides the use of peptide uroaktivin for a more efficient therapy of diseases or conditions for which thrombolytic medicines are indicated. These conditions are especially the following: acute coronary thrombosis (acute myocardial infarction), acute ischemic brain infarction, acute lung thromboembolism, deep venous thrombosis, acute arterial thromboembolism, acute arterial thrombosis. The peptide uroaktivin would as well be used for assurance of permeability of arteriovenous cannulas and intravenous catheters.

Description

PEPTIDE UROAKTIVIN, AS AN ACTIVATOR OF THE ENZYME UROKINASE
FIELD OF THE INVENTION
The present invention relates to the peptide uroaktivin, that binds to the urokinase type plasminogen activator (urokinase) and as a consequence of binding increases the enzymatic activity of urokinase. In particular, the present invention relates to the use of said peptide for the therapy of conditions and diseases, that are usually treated with recombinant tissue type or urokinase type plasminogen activator, i.e. all the indications that require thrombolytic medicines. The present invention relates in general to thrombolytic medicines, especially to urokinase type plasminogen activator.
BACKGROUND OF THE INVENTION
The unwanted formation of coagulation clots inside blood vessels (thrombosis) is one of the main cardiovascular complications. The formation of a coagulation clot (thrombus) may provoke a decrease or complete interruption of blood flow through the vessel. The interruption of blood flow may lead to an acute myocardial infarction, ischemic brain infarction, venous thromboembolism or lung embolism. Antithrombotic medicines are used for the prevention of clot formation, among which there are anticoagulants (vitamin K antagonist), inhibitors of thrombocyte aggregation (acetylsalicylic acid, heparin and heparin derivatives, hirudin, clopidogrel, dipiridamol) and thrombolytics (fibrinolytics). Thrombi in veins, where the blood flow is slower, are composed mainly of a fibrin network and to a less extent contain thrombocytes, therefore, primarily, the therapy with anticoagulants of the warfarin and heparin type is used. Thrombi in arteries, where the blood flow is faster, contain more thrombocytes and less fibrin. For this reason, acetylsalicylic acid is used for prophylaxis of arterial thrombi or in case of occlusion - thrombolytic medicines. Anticoagulants and inhibitors of thrombocyte coagulation prevent the formation of new and the enlargement of present thrombi, while thrombolytic medicines are adopted for the immediate break-up (lysis) of obstructive clots, but they do not prevent the formation of new thrombi. Physiologic fibrinolysis
Fibrinolysis is a normal process in the body, that is going on simultaneously with coagulation and preserves homeostasis by preventing the formation of thrombi, that would obstruct the flow of blood. During the process of fibrinolysis, fibrinogen is transformed by thrombin to fibrin monomers, that polymerize and form protofibirls, composed of two antiparallel non- covalently linked chains. Inside each chain, the fibrin monomers are linked together covalently. The main fibrinolytic enzyme is the serine protease plasmin, which is generated from plasminogen by activators. Plasminogen contains secondary structure motifs that specifically bind lysine and arginine of fibrin(ogen) which enables plasminogen to bind to the network of fibrin during thrombus formation. In the process of fibrinolysis, plasminogen is transformed into its active form and cleaves fibrin on the C-terminal end in regard to the bound lysines and arginines. During its activity, plasmin initially creates gaps in the fibrin network with further degradation resulting in the complete solubilisation of fibrin (Walker, 1999).
The primary activators of plasminogen are the tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA, urokinase). tPA is released from endothelial cells of blood vessels, while urokinase is released from fibroblasts, endothelial cells and monocytes.
The process of fibrinolysis is regulated by many inhibitors (inhibitor of plasminogen activator-1 and -2, PAI-I, PAI-2; alpha2-antiplasmin; alfa2-macroglobulin; serpin; TAFI), which enable the localisation of the lysis.
Thrombolytic medicines and their indications
Fibrinolysis can be triggered pharmacologically with the so called thrombolytic (fibrinolytic) therapy. The most commonly used thrombolytics for systemic use are:
- alteplase (recombinant tPA)
- anistreplase
- streptokinase and
- urokinase.
Besides them, other thrombolytics are sometimes used:
- staphilokinase (bacterial recombinant protein)
- duteplase (tPA composed from two protein chains) - reteplase (a tPA-similar protein with a longer half time of elimination) and
- tenecteplase (tPA with two modified amino acids).
Thrombolytic medicines are applied parenterally, due to their protein nature. The most important aspect of thrombolytic therapy is that it must be based on the individual characterisation of the thrombotic complication, the individual's status and his/her anamnesis. During the use of thrombolytics, it must be taken into consideration the balance between their potential positive effects and the risk of bleeding, which is the main adverse effect of thrombolysis.
Thrombolytics are used for the treatment of:
- acute coronary thrombosis (acute myocardial infarction)
- acute ischemic brain infarction
- acute ling thromboembolism
- deep venous thrombosis
- acute arterial thromboembolism
- acute arterial thrombosis.
Thrombolytics are used for ensuring the permeability of:
- arteriovenous cannulas
- intravenous catheters.
Urokinase and its action
Urokinase is a 411 amino acid long protein composed of three domains: C-terminal catalytical domain with the active serine protease site, N-terminal Kringle domain and the epidermal growth factor domain, that binds to the uPAR/CD87 receptor. Urokinase is synthesized in the form of a zymogen (prourokinase - pro-uPA and single-chain urokinase - sc-uPA) and is activated by proteolytic cleavage of the Lys[158]-Ile[159] bond. The two resulting chains remain connected via a disulphide bond.
Urokinase directly cleaves the peptide bond between amino acids Arg[560]-Val[561] of plasminogen to produce plasmin that in turn activates the fibrinolytic system. Plasmin cleaves fibrin, fibrinogen in other plasma proteins in coagulation clots, among others the coagulation factors V and VIII. Single- and double-chain forms of urokinase bind to receptors on the plasma membrane: uPAR/CD87 and receptors of the LDL receptor family: LDLR-relative protein, receptor alfa2-macroglobuline (LRP/α2-MR) and VLDL- receptor. The affinity of the urokinase binding to the receptors of the LDL receptor family (IQ- (l -2)-10~8 M) is about one degree of magnitude lower than the binding affinity to uPAR/CD87. Moreover, urokinase can bind to other receptors through its protease and Kringle domain. The multidomain structure of urokinase enables its polyfunctional activity, as it contains many binding sites for substrates and other cell receptors.
Tissue type plasminogen activator and its action
Tissue type plasminogen activator (tPA) is a serine protease and like urokinase, a member of the SlA serine protease family. It is normally present on the surface of endothelial cells of veins, capillaries, lung arteries, heart and uterus and is released when the vessel is damaged. tPA is synthesized as a single-chain polypeptide which is converted into a double-chain polypeptide connected with a disulphide bond, after the action of plasmin or trypsin (cleavage of the Arg-Ile bond). The heavy chain of tPA represents the N-terminal part (Mr 39.000), while the light chain represents the C-terminal end (Mr 33.000). tPA is composed of five distinct structural domains: the domain homologous to the fibronectin finger-like structure, domain homologous to the epidermal growth factor, active serine protease site and Kringle domains 1 and 2 (as they are present in urokinase, plasminogen, thrombin and factor XII). All but the active site are situated on the heavy chain of tPA. tPA converts plasminogen into plasmin. The specificity of the activation depends on the affinity of tPA towards fibrin and the formation of a tertiary complex between tPA, plasminogen and fibrin, which results in the strong increase of the activational rate of plasminogen on the fibrin surface. The functional domains of tPA, that are responsible for the affinity towards fibrin and the increase of tPA activity are situated inside the N-terminal region (heavy chain), probably in the finger-like domain (Banyai, 1983) and the Kringle domain 2 (van Zonneveld, 1986).
The role of cytokeratins in plasminogen activation
Activation of plasminogen on the cell surface is much faster compared to the activation in solution, as the interaction of plasminogen with binding proteins results in a conformational change of plasminogen to a conformation that is more open for proteolytic action (Ellis, 1991).
Cytokeratin 8 is the main binding protein of plasminogen on the membrane of breast cancer cells. Cytokeratin 8 is an intermediate filament protein, which pairs with cytokeratin 18 and forms the insoluble structural cytoplasmic skeleton. The C-terminal part of cytokeratin 8 penetrates through the cell membrane (Hembrough, 1995; Ditzel, 1997) and binds plasminogen (Kralovich, 1998). The activation of plasminogen is accelerated by the binding of tPA to cytokeratin 8 or to cytokeratin 18 (Kralovich, 1998).
The sequence of peptide uroaktivin is derived from the highly homologous (although not identical) sequence of the C-terminal part of cytokeratins 1, 2, 8, 10 and 18. We suggest that this region is responsible for the binding of tPA and urokinase.
State of the art
The state of the art in the field of usage of urokinase for the break down of unwanted coagulation clots in the body (fibrinolysis) are described in the following references (to our present knowledge, activators of urokinase have not yet been patented):
- Banyai L, Varadi A, Patthy L. Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator. FEBS Lett. 1983;163(1):37-41.
- van Zonneveld AJ, Veerman H, Pannekoek H. On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid. J Biol Chem. 1986, 261(30):14214-8.
- Ditzel H, Garrigues U, Andersen C, Larsen M, Garrigues H, Svejgaard A, Hellstrδm I, Hellstrόm K, Jensenius J: Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment. Proc Natl Acad Sci U S A 1997, 94(15):8110-81 15.
- Ellis V, Behrendt N, Danø 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 external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines. J Cell Sci 1995, 108 ( Pt 3):1071-1082.
- Kralovich KR, Li L, Hembrough TA, Webb DJ, Karns LR, Gonias SL. Characterization of the binding sites for plasminogen and tissue-type plasminogen activator in cytokeratin 8 and cytokeratin 18. J Protein Chem. 1998;17(8):845-54.
- Walker JB, Nesheim ME. The 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;274(8):5201-12.
- United States Patent 6,861,054; Higazi. suPAR stimulating activity of tcuPA-mediated fibrinolysis and different uses thereof. 2005.
- United States Patent 4,996,050; Tsukada, et al.: Fibrinolytic activity enhancer. 1991.
DESCRIPTION OF FIGURES
Fig. 1 shows the results of the binding of uroaktivin to the urokinase type plasminogen activator acquired by the surface plasmon resonance technique. The curves represent the binding of urokinase type plasminogen activator to the immobilised uroaktivin and its dissociation at concentrations 10 nM, 20 nM and 50 nM (in this order from bottom up).
Fig. 2 shows the activation of plasminogen in the presence of peptide uroaktivin. The activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was measured after a period of 90 min.
Fig. 3 shows the kinetics of the activation of plasminogen in the presence of peptide uroaktivin. The activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was followed for 100 min. Fig. 4 shows the activation of plasminogen in the presence of peptide uroaktivin. The activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM tissue type plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was measured after a period of 90 min.
Fig. 5 shows the kinetics of the activation of plasminogen in the presence of peptide uroaktivin. The activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM tissue type plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was followed for 100 min.
Fig. 6 shows the control experiment of the activation of plasminogen in the presence of peptide uroaktivin and in the absence of plasminogen activator (uPA or tPA). The activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of peptide. The fluorescence was measured after a period of 90 min.
Fig. 7 shows the control experiment of the activation of plasminogen in the presence of a negative control undekapeptide. The activation of plasminogen was determined by measuring the fluorescence at 470 nm in the presence of 500 pM urokinase plasminogen activator, 500 nM plasminogen, specific substrate for plasmin D-Ala-Lev-Lys-AMC (0.5 mM) and increasing concentrations of undekapeptide. The fluorescence was measured after a period of 90 min.
Fig. 8 shows the experiment of lysis of fibrin clot in the presence of peptide uroaktivin. The lysis of fibrin clot was determined by measuring the absorbance at 405 nm in the presence of 500 nM plasminogen, urokinase plasminogen activator (250 pM, 500 pM and 1 nM) and increasing concentrations of uroaktivin. Absorbance was measured until the total lysis of the fibrin clot. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the amino acid sequence in uroaktivin (peptide with amino acid sequence SEQ ID NO:1) and peptidomimetics, deduced from the amino acid sequence of uroaktivin.
Peptidomimetics, within the context of this application can be understood as being peptides which are 50, 70, 80, 90, 95, 98, 99, or 99.9% identical to SEQ ID NO:1. Peptidomimetics preferably have the same biochemical function as has SEQ ID NO: 1. The invention thus also relates to peptides with are 50, 70, 80, 90, 95, 98, 99, or 99.9% identical to SEQ ID NO:1. These peptides preferably have the same biochemical function as has SEQ ID NO:1. Peptides of the invention are preferably 8, 9, 10, 11, 12, 13, 15, 20, 30, 50, 100 amino acids in length.
Within the context of the present invention, the "% identity" of a protein relative to a reference protein of a defined length shall be understood as follows: A peptide that is 50 percent identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide, which is 50 percent identical to the reference polypeptide over its entire length. Of course, other polypeptides will meet the same criteria. The term "sequence identity" as used herein means that the sequences are compared as follows. The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default (BLOSUM62) matrix (values -4 to +11) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (per each additional consecutive null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference polypeptide.
The invention also relates to DNA coding for the peptides of the invention.
The present invention further relates to pharmaceutical composition comprising uroaktivin or peptidomimetics, deduced from the amino acid sequence of uroaktivin. In a preferred embodigment, the pharmaceutical compositions contain uroaktivin or peptidomimetics, deduced from the amino acid sequence of uroaktivin. Preferably, the pharmaceutical composition are suitable for parenteral application.
The invention further relates to medical equipment, designated to contain uroaktivin or peptidomimetics of the invention, which can be used in direct or indirect prevention of blood clotting, in particular in application of catheters and stents.
The invention further relates to the use of peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin, as activator of enzymes, which accelerate the process of fibrinolysis.
The invention further relates to the use of eptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics deduced from the amino acid sequence of uroaktivin as an activator of urokinase type plasminogen activator.
The invention further relates to the use of peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics deduced from the amino acid sequence of uroaktivin as activator of tissue type plasminogen activator.
The invention further relates to peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin to be used as a drug. The invention further relates to the use of said peptides as a drug.
The invention further relates to a pharmaceutical composition, characterised in that it contains peptide uroaktivin with amino acid sequence SEQ ID NO.1 and/or peptidomimetics, deduced from the amino acid sequence of uroaktivin, to be used as activator of enzymes, which accelerate the process of fibrinolysis, as activator of urokinase type plasminogen activator and/or as activator of tissue type plasminogen activator.
The invention further relates to the use of peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin for preparation of pharmaceutical compositions for treatment of diseases and conditions wherein thrombolytics are indicated.
The application further relates to the use of peptide uroaktivin with amino acid sequence SEQ ID N0.1 and peptidomimetics, deduced from the amino acid sequence of uroaktivin, for preparation of pharmaceutical compositions for treatment of one of acute coronary thrombosis (acute myocardial infarction), acute ischemic brain infarction, deep venous thrombosis, acute lung thromboembolism, acute arterial thromboembolism, acute arterial thrombosis, and for assurance of permeability of arteriovenous cannulas and intravenous catheters.
EXAMPLES
Hereinafter, the present invention is illustrated in more detail and specifically with reference to the Examples, which however are not intended to limit the present invention.
Example 1. The design of the peptide
The amino acid sequences of cytokeratins 1, 2, 8, 10 in 18 were derived from the NCBI Protein Database (entry # 1346343, 547754, 225702, 40354192 and 30311, respectively). The sequences were aligned with the ClustalW multiple sequence alignment software (www.ch.embnet.org) and on the basis of the aligned sequences, the amino acid sequence of the peptide uroaktivin was designed. The resulting amino acid sequence does not belong to neither cytokeratins, and does not belong to any of the known living organisms according to the Blast data base (http://blast.ncbi.nlm.mh.gov/Blast.cgi)
Example 2. The synthesis of the peptide
The peptide uroaktivin with the amino acid sequence SEQ ID NO: 1 was synthesized at Biosynthesis Inc. (Denver, TX, USA). The purity of the synthesized peptide was 85.6 % determined by HPLC and mass analysis.
Example 3. Testing or the binding of urokinase type plasminogen activator to uroaktivin with the method of surface plasmon resonance The binding of urokinase to uroaktivin was determined with a Biacore X system (Biacore, Sweden). The experiment was carried out on a CM5 sensor chip (cat. # BR-1003-98 Biacore). Uroaktivin was immobilised at a constant flow of 1 μL/min for 10 minutes on the flow cell #2 (up to 300 RU). The flow cell was then washed with 5 μL 10 mM glycine buffer (pH 2.2) at a flow of 30 μL/min. The flow cell # 1 was used as a reference cell. The same washing procedure was used for the regeneration of the uroaktivin surface between injections of urokinase. uPA was dissolved in a PBST buffer (PBS, pH 7.4 with 0.05% Tween 20). The immobilised uroaktivin was treated with different concentrations of urokinase: 10 nM, 20 nM and 50 nM. All the measurements were done at 250C and at a flow of 1 μL/min in 1% PBST buffer. 5 μL of uPA was injected at each measurement. The association and dissociation curves of urokinase type plasminogen activator on/off the peptide uroaktivin are shown on Fig. 1. With the drifting baseline method of the Biacore evaluation software, we determined a Ka of 9.8 nM .
Example 4. Plasminogen activation
The increased activation of plasminogen in the presence of peptide uroaktivin was determined by measuring fluorescence at 470 nm, and matches the increased plasmin formation. Final concentrations of 500 pM urokinase type or tissue type plasminogen activator, 500 nM plasminogen and 0.5 mM specific substrate for plasmin D-Ala-Lev-Lys-AMC were used in the test. The amount of generated plasmin in 90 min and the kinetics of its generation show a strongly increased generation in the presence of peptide uroaktivin. The formation of plasmin depends on the concentration of peptide uroaktivin (Fig. 2-5). Plasmin was not generated in control experiments, where plasminogen activator was not present.
uPA
Urokinase type plasminogen activator (uPA) (50OpM) was dissolved in phosphate buffer, pH 7.4 and added to a 50 μL well. Uroaktivin was added in concentrations of 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM and 5 μM and incubated at 370C for 30 min. 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 into microtiter wells and the generation of plasmin was followed for 1.5 hours at 37°C (Ex = 370 nm and Em = 470 nm) with a fluorescence microtiter plate reader (Safire 2, Tecan). In a control experiment an undekapeptide or a dodekapeptide with a different sequence were used. A control experiment without uPA added was also employed. The result is representative of three independent experiments. The average values ± standard deviation of four parallels are given. tPA
Tissue type plasminogen activator (tPA) (50OpM) was dissolved in phosphate buffer, pH 7.4 and added to a 50 μL well. Uroaktivin was added in concentrations of 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM and 5 μM and incubated at 370C for 30 min. 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 into microtiter wells and the generation of plasmin was followed for 1.5 hours at 37°C (Ex = 370 nm in Em = 470 nm) with a fluorescence microtiter plate reader (Safire 2, Tecan). In a control experiment an undekapeptide or a dodekapeptide with a different sequence were used. A control experiment without tPA added was also employed. The result is representative of three independent experiments. The average values ± standard deviation of four parallels are given.
Example 5. Fibrin clot lysis test
The fibrin clot was prepared with the addition of 50 μL human thrombin (0.3 U/mL, final concentration) (INIH U = 0.324 ± 0.073 μg of thrombin) and CaCl2 (20 mM) to 50 μL of human fibrinogen (2 mg/niL, final concentration) in a 96-well microtiter plate. The formation of the clot was carried on for 3 hours at room temperature. 50 μL of 1 μM plasminogen and 50 μL of urokinase plasminogen activator (500 pM, 1.0 nM, 2.0 nM) with uroaktivin (20 μM, 10 μM, 5 μM, 0 M) were than added. Changes in the opacity of the clot were followed by measuring the absorbance at 405 nm at 25 0C with a microtiter plate reader. The measurements were done in four parallels and repeated three times.

Claims

1. Amino acid sequence in uroaktivin (peptide with amino acid sequence SEQ ID NO.l) and peptidomimetics, deduced from the amino acid sequence of uroaktivin.
2. Pharmaceutical composition, designated to contain uroaktivin or peptidomimetics, deduced from the amino acid sequence of uroaktivin.
3. Pharmaceutical composition, characterised in that it contains uroaktivin or peptidomimetics, deduced from the amino acid sequence of uroaktivin, according to claims 1-2, which enables parenteral application.
4. Medical equipment, designated to contain uroaktivin or peptidomimetics, deduced from the amino acid sequence of uroaktivin, according to claims 1 -2, which is used in direct or indirect prevention of blood clotting, in particular in application of catheters and stents.
5. Peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin according to claim 1 , to be used as activator of enzymes, which accelerate the process of fibrinolysis.
6. Peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin according to claim 1 , to be used as activator of urokinase type plasminogen activator.
7. Peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin according to claim 1 , to be used as activator of tissue type plasminogen activator.
8. Peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin according to claim 1 , to be used as a drug.
9. Pharmaceutical composition, characterised in that it contains peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin according to claim 1, to be used as activator of enzymes, which accelerate the process of fibrinolysis, as activator of urokinase type plasminogen activator and as activator of tissue type plasminogen activator.
10. The application of peptide uroaktivin with amino acid sequence SEQ ID NO. l and peptidomimetics, deduced from the amino acid sequence of uroaktivin according to claim 1 , for preparation of pharmaceutical compositions for treatment of diseases and conditions wherein thrombolytics are indicated.
11. The application of peptide uroaktivin with amino acid sequence SEQ ID NO.l and peptidomimetics, deduced from the amino acid sequence of uroaktivin according to claim 1, for preparation of pharmaceutical compositions for treatment of acute coronary thrombosis (acute myocardial infarction), acute ischemic brain infarction, deep venous thrombosis, acute lung thromboembolism, acute arterial thromboembolism, acute arterial thrombosis, as well as for assurance of permeability of arteriovenous cannulas and intravenous catheters.
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