Patent Application
20050058636
Subject of this invention is a medicament against thrombotic diseases which contains as an active ingredient a polypeptide which is derived from the domain-5 of kininogen.
It is known that after vascular injury in patients, pathological thrombosis is the critical event leading to acute myocardial infarction, unstable angina pectoris or stroke. Under physiological conditions, primary haemostasis after vascular injury includes adhesion of platelets to the exposed extracellular matrix in the subendothelium with subsequent platelet aggregation. At high shear rates platelets predominantly adhere to von Willebrand factor and collagen, whereas under low shear or static conditions several other extracellular matrix proteins, including fibrin or vitronectin, mediate initial platelet adhesion via specific interactions between the Arg-Gly-Asp (RGD) sequence of the adhesive matrix proteins and specific adhesion receptors of the αIIbβ3-integrin type expressed on platelets.
The temporary platelet thrombus serves as activation surface for the blood coagulation system, culminating in the production of fibrin to seal and stabilize the wound site. A balance between pro-coagulatory and anti-coagulatory factors as well as the contribution of the fibrinolytic system serves to regulate thrombus formation and finally leads to clot dissolution. Tissue-type plasminogen activator (tPA) is the primary protease, which activates plasminogen in a fibrin-specific manner. Subsequently, the formed enzyme plasmin initiates intravascular fibrinolysis. Regulation of plasminogen activation in a fibrin-specific fashion is achieved by plasminogen activator inhibitor-1 (PAI-1), which forms a stable inactive complex with tPA, thereby preventing the initial thrombus against extensive lysis. PAI-1 becomes functionally stabilized by high affinity complex formation with vitronectin, an abundant plasma and wound matrix-associated adhesive glycoprotein, which is also contained together with PAI-1 in platelet α-granules. The binding of PAI-1 to vitronectin occurs primarily within the amino-terminal region of the adhesive protein. This region of vitronectin also contains binding sites for integrins, the urokinase receptor and kininogen. An elevated PAI-1 level constitutes an important thrombotic risk factor for e.g. myocardial infarction or deep venous thrombosis due to an overall increased antifibrinolytic potential.
HK (=high molecular weight kininogen) which is present in plasma at a concentration of 0.67 μM, was initially identified as a non-enzymatic cofactor in the initiation of the contact phase system. HK appears to be associated with vascular injury, inflammation or activation of complement in humoral immune defense. In particular, the enzyme kallikrein liberates the short-lived vasodilator peptide bradykinin from HK domain 4 thereby generating two chain kinin-free HKa. Domain 5 in HKa is rich in His, Gly, and Lys, enabling HKa to bind to anionic surfaces, zinc or heparin. HK/HKa interacts with a number of binding proteins on cell surfaces including the binding protein for globular C1q (gC1qR), urokinase receptor and cytokeratin-1 on endothelial cells, as well as glycoprotein Ib and cell-associated thrombospondin on platelets. It has already been found that HKa and particularly domain 5 bind directly to vitronectin, thereby competing for leukocyte as well as endothelial cell adhesion. Moreover, it has also been previously demonstrated that HKa and PAI-1 compete for proximal binding sites within the amino-terminal domain of vitronectin. Finally, domain 5 of HK binds to the β2-integrin Mac-1 on granulocytes, thereby regulating the Mac-1-dependent leukocyte recruitment in vivo.
Experimental evidence from in vitro and vivo studies renders HK an anti- rather than a prothrombotic agent. A prothrombotic phenotype was reported for kininogen deficient rats, and patients deficient in kininogen or other proteins of the contact phase system are at increased risk for thrombosis:
Based on these in vitro and in vivo properties of kininogen it has been examined whether polypeptides of this endogenous factor are useful in the acute as well as in the prophylactic therapy in vascular diseases and may be valuable as antithrombotic drugs.
It has now been found a medicament against thrombotic diseases which contains as active ingredient the domain-5 of high molecular weight kininogen or a domain-5-peptide of the carboxy-terminal region of the high molecular weight kininogen. Preferred is a medicament wherein the domain-5-peptide is the HK [His-483-Lys502]-peptide whose sequence may be seen from the SEQ ID No.1 of the attached sequence listing. Also, peptides derived from this HK-sequence may be used advantageously if they exhibit antithrombotic efficacy.
Such medicaments for parenteral administration in a therapeutically effective amount, i.e. 0.1-20 mg/kg bodyweight may be used in patients with arterial thrombosis, acute myocardial infarction or lung fibrosis. Moreover, due to their pro-fibrinolytic potential they are valuable to decrease the lysis resistance of unwanted thrombi. Finally, the domain-5-peptides are save and likely non-immunogenic, since they are substances which are derived from endogenous plasma proteins.
The efficacy of domain-5-peptides in said medicaments is based on the scientific finding that another macromolecular ligand for vitronectin, the primary inhibitor of fibrinolysis PAI-1, binds with high affinity to the aminoterminal portion of the adhesive protein within the neighbourhood of binding sites for integrins, urokinase receptor and HK. Thus, PAI-1 does not only constitute another anti-adhesive factor for integrin- and urokinase receptor-mediated cell adhesion to vitronectin, but also competes with activated, two-chain HK (HKa) for interaction with vitronectin. Vice versa, domain 5 of HK and derived polypeptides thereof can efficiently block the binding of PAI-1 to vitronectin or displace the inhibitor from its complex with the adhesive protein, respectively. Since only in complex with vitronectin the PAI-1 function can be stabilized predominantly in the vascular extracellular matrix, dissociation of PAI-1 from vitronectin is associated with the rapid decay of its inhibitor function. Thus, at sites of codistribution of PAI-1 and HKa, the latter factor is able to dissociate the inhibitor from binding to vitronectin and thereby HKa can lower the antifibrinolytic activity of PAI-1. Consequently, HKa and polypeptides thereof may function as a new type of PAI-1 inhibitor and would thus be of therapeutic interest, since increased levels of PAI-1 in patients correlate with an elevated anti-fibrinolytic activity or an increased resistance to thrombolysis. These latter relationships have been evaluated and validated in several clinical studies. Due to the efficiency of kininogen polypeptides to serve as PAI-1 inhibitor, both the anti-fibrinolytic activity as well as the thrombolytic resistance would decrease thereby elevating the net fibrinolytic capacity.
The efficacy of the domain-5-peptide against thrombotic diseases is further increased if they are administered in combination with urokinase or tissue plasminogen activator (tPA) as they eliminate PAI-1. Such a combination medicament is a very powerful agent against thrombotic complications.
The PAI-1 inhibitor activity of kininogen polypeptides may be demonstrated by the following examples:
The extracellular matrix of human umbilical vein endothelial cells was prepared according to standard procedures. PAI-1-depleted extracellular matrix was obtained by subsequent incubation with glycine (0.1 M, pH 2.3) for 2 h at 25° C. Wells were washed and blocked with phosphate buffered saline containing 3% (wt/vol) bovine serum albumin before proceeding with binding assays. Binding of PAI-1 (2 μg/ml) to the extracellular matrix was performed in a final volume of 50 μl Tris-buffered saline containing 0.1% Tween-20, 0.3% (wt/vol) bovine serum albumin for 2 h at 22° C. in the absence or presence of 10 ∞M ZnCl2 and different kininogen forms. Following the incubation period and a washing step, bound PAI-1 was quantitated by anti-human PAI-1 monoclonal antibody 12A4, followed by addition of 1:1000 diluted peroxidase-conjugated goat anti-mouse immunoglobulins. The conversion of the substrate 2,2,-azino-di(3-ethyl) benz-thiazoline sulphate was monitored at 405 nm in a Thermomax microtiter plate reader. Nonspecific binding of PAI-1 to bovine serum albumin-coated wells was used as blank and was subtracted to calculate the specific binding.
Plasminogen Activation Assays:
PAI-1 (0.1-40 nM) or buffer alone were incubated without or together with multimeric vitronectin (1 μg/ml) in the absence or presence of different kininogen forms and for different time periods (as indicated in the figure legends) in Tris-buffered saline containing 0.1% (wt/vol) Tween-20 and 0.1% (wt/vol) bovine serum albumin at 37° C. After different time intervals, aliquots of reaction mixtures were transferred into microtiter wells and urokinase was added. After an incubation period of 30 min at 22° C., a mixture of plasminogen (final concentration 0.22 μM) and the chromogenic substrate S-2251 (0.8 mM) was added, and the rate of plasmin generation was followed at 405 nm.
In addition to its role as anti-adhesive factor for several cell types, domain 5 of HK was also able to block the adhesion of platelets to vitronectin-rich wound matrix. Moreover, in a concentration-dependent fashion the aggregation of platelet-rich plasma or the formation of a blood platelet fibrin clot, respectively, was blocked in a concentration dependent fashion by HKa, the domain 5 of HK or polypeptides thereof. All these activities of HK are observed within a range of concentrations of the protein or the fragments, respectively, which are within the physiological levels of the plasma protein.
Microtiter plates were coated with 5 μg/ml αIIbβ3-integrin in Tris-buffered saline, pH 7.4, containing 1 mM CaCl2, 1 mM MgCl2 overnight at 4° C. and then blocked with 3% (w/v) bovine serum albumin. Binding of multimeric vitronectin (2 μg/ml) was performed in a final volume of 50 μl Tris-buffered saline containing 0.1% Tween-20, 0.3% (w/v) bovine serum albumin, 1 mM CaCl2 and 1 mM MgCl2 for 2 h at 22° C. in the absence or presence of 10 μM ZnCl2 and different kininogen forms. After the incubation period, monoclonal anti-vitronectin antibody VN7 at a concentration of 125 ng/ml was added, followed by addition of 1:1000 diluted peroxidase-conjugated goat anti-mouse immunoglobulins, respectively. Binding was quantitated as described above.
Platelet Adhesion onto Vitronectin-Coated Plates:
Multiwell plates were coated with 5 μg/ml multimeric vitronectin (dissolved in bicarbonate buffer, pH 9.6) and blocked with 3% (wt/vol) bovine serum albumin for 16 h at 4° C. Platelets stained with calcein acetoxymethyl ester (calcein-AM, 2.5 μM) (15 min at 37° C. in the dark) at a density of 5×106 cells suspended in 50 μl HEPES-Tyrode buffer containing 4 mM MgCl2 were added together with the respective competitors and incubated for 30 min at 37° C. After three washes with phosphate buffered saline, bound platelets were lysed with 100 μl of lysis buffer (150 mM NaCl, 10 mM Tris-CH1, pH 7.5, 5 mM EDTA, 1% (wt/vol) NP-40, 1 mM phenyl-methylsulfonyl fluoride, 20 μg/ml soybean trypsin inhibitor) and quantified in a fluorescent plate reader (excitation filter 492 nm, emission filter 535 nm).
Aggregation of Platelet-Rich Plasma:
Platelet-rich plasma from fresh whole blood in sodium citrate was prepared by centrifugation of anticoagulated whole blood at 150 g for 20 min. After incubation of 0.45 ml of platelet rich plasma with different kininogen forms as indicated in the figure legends, and addition of 5 μl of adenosine-triphosphate at a final concentration of 5 μM, platelet aggregation was induced. Aggregation was quantitated by following the change in light transmission in a standardized aggregometer.
The examples for the treatment with kininogen polypeptides may be summarized as follows:
The attached
| Number | Date | Country | Kind |
|---|---|---|---|
| 02005370.8 | Mar 2002 | EP | regional |
