AU2013202565A1

AU2013202565A1 - Therapeutic application of Kazal-type serine protease inhbitors

Info

Publication number: AU2013202565A1
Application number: AU2013202565A
Authority: AU
Inventors: Kay Hofmann; Ulrich Kronthaler; Stefan Schmidbauer; Stefan Schulte; Thomas Weimer
Original assignee: CSL Behring GmbH Deutschland
Current assignee: CSL Behring GmbH Deutschland
Priority date: 2007-02-12
Filing date: 2013-04-05
Publication date: 2013-05-02

Abstract

The subject of the present invention is, in the most general aspect, the therapeutic application of the Kazal-type serine protease inhibitor Infestin or domains thereof or modified Kazal-type serine protease inhibitors based on Infestin homologs, which prevent the formation and/or stabilization of three-dimensional arterial or venous thrombi by interfering with proteins involved in activation of the so-called intrinsic coagulation pathway. In particular the present invention relates to the use of said Kazal-type serine protease inhibitors or fragments thereof or modified Kazal-type serine protease inhibitors, in the treatment or prophylaxis of a condition or disorder related to arterial thrombus formation, i.e. stroke or myocardial infarction, inflammation, complement activation, fibrinolysis, angiogenesis and/or diseases linked to pathological kinin formation such as hypotonic shock, edema including hereditary angioedema, bacterial infections, arthritis, pancreatitis, or articular gout, Disseminated Intravasal Coagulation (DIC) and sepsis,

Description

5 Therapeutic application of Kazal-type serine protease inhibitors The subject of the present invention is, in the most general aspect, the therapeutic application of the Kazal-type serine protease inhibitor Infestin or domains thereof or modified Kazal-type serine protease inhibitors based on Infestin homologs, which 10 prevent the formation and/or stabilization of three-dimensional arterial or venous thrombi by interfering with proteins involved in activation of the so-called intrinsic coagulation pathway. In particular the present invention relates to the use of said Kazal-type serine protease inhibitors or fragments thereof or modified Kazal-type serine protease inhibitors, in the treatment or prophylaxis of a condition or disorder 15 related to arterial thrombus formation, i. e. stroke or myocardial infarction, inflam mation, complement activation, fibrinolysis, angiogenesis and/or diseases linked to pathological kinin formation such as hypotonic shock, edema including hereditary angioedema, bacterial infections, arthritis, pancreatitis, or articular gout, Dissemi nated Intravasal Coagulation (DIC) and sepsis. 20 Vessel wall injury triggers sudden adhesion and aggregation of blood platelets, followed by the activation of the plasma coagulation system and the formation of fibrin-containing thrombi, which occlude the site of injury. These events are crucial to limit post-traumatic blood loss but may also occlude diseased vessels leading to 25 ischemia and infarction of vital organs, In the waterfall model, blood coagulation proceeds by a series of reactions involving the activation of zymogens by limited proteolysis culminating in generation of thrombin, which converts plasma fibrinogen to fibrin and activates platelets. In turn, collagen- or fibrin-adherent platelets facili tate thrombin generation by several orders of magnitude via exposing procoagulant 30 phospholipids (mainly phosphatidyl serine) on their outer surface, which propagates assembly and activation of coagulation protease complexes and by direct interaction between platelet receptors and coagulation factors. Two converging pathways for coagulation exist that are triggered by either extrinsic (vessel wall) or intrinsic (blood-borne) components of the vascular system. The 5 "extrinsic" pathway is initiated by the complex of the plasma factor VI (FVII) with the integral membrane protein tissue factor (TF), an essential coagulation cofactor that is absent on the luminal surface but strongly expressed in subendothelial layers of the vessel and which is accessible or liberated via tissue injury. TF expressed in circulating microvesicles might also contribute to thrombus propagation by sustain 10 ing thrombin generation on the surface of activated platelets. The "intrinsic" or contact activation pathway is initiated when factor XII (FXII, Hageman factor) comes into contact with negatively charged surfaces in a reaction involving high molecular weight kininogen and plasma kallikrein. FXII can be 15 activated by rnacromolecular constituents of the subendothelial matrix such as glycosaminoglycans and collagens, sulfatides, nucleotides and other soluble polyanions or non-physiological material such as glass or polymers. One of the most potent contact activators is kaolin and this reaction serves as the mechanistic basis for the major clinical clotting test, the activated partial thromboplastin time 20 (aPTT), which measures the coagulation capacity via the "intrinsic" pathway. In reactions propagated by platelets, activated FXII then activates FXI to FXia and subsequently FXIa activates factor IX. The complex of FVIIa, which FVIllIa has been previously activated by traces of FXa and/or Thrombin, and FIXa (the tenase complex) subsequently activates FX (see figure 1, "left arm"). Despite its high 25 potency to induce blood clotting in vitro, the (patho) physiological significance of the FXII-triggered intrinsic coagulation pathway is questioned by the fact that hereditary deficiencies of FXII as well as of high molecular weight kininogen and plasma kallikrein are not associated with bleeding complications. Together with the obser vation that humans and mice lacking extrinsic pathway constituents such as TF and 30 FVII suffer from severe bleeding this has led to the current hypothesis that for the cessation of bleeding in vivo exclusively the extrinsic cascade is required

Claims

1. Mutant Kazal inhibitors derived from SPINK-1, wherein said inhibitors are mutated in order to increase homology to Infestin -4 and which are suitable for 10 preventing formation and/or stabilization of three-dimensional arterial or venous thrombi.

2. Mutant Kazal inhibitors according to claim 1, wherein the contact sites with the inhibited protease are derived from domain 4 of Kazal-type inhibitor 15 Infestin.

3. Mutant Kazal inhibitors according to claim 2, wherein additional amino acid positions within the modified Kazal inhibitor sequence are derived from domain 4 of Infestin. 20

4. Mutant Kazal inhibitors according to any of the preceding claims, which are SPINK K1, K2 and K3 (SEQ ID NO 2, 3 and 4).

5. Mutant Kazal inhibitors according to any of the preceding claims and/or 25 Infestin or fragments thereof, preferably Infestin 3-4 or more preferred Infestin-4, which inhibitors are linked to half-life enhancing polypeptides.

6. The inhibitors of claims 1 to 5 characterized in that the half-life enhancing polypeptide is derived from a member of the human albumin protein family, namely 30 albumin, afamin, alpha-fetoprotein or vitamin D binding protein. 45;

7. The inhibitors of claims 1-5 characterized in that the half-life enhancing polypeptide is human albumin or a variant or domain or part thereof.

8. The inhibitors of claims 1-5 characterized in that the half-life enhancing 5 polypeptide is an immunoglobulin or a part thereof.

9. The inhibitors of claim 8 characterized in that the immunoglobulin part is an IgG Fc portion.

10 10. The inhibitors of claims 5 to 9 characterized that the half-life enhancing polypeptides are linked to the Kazal inhibitor moiety via linkers, preferably cleavable linkers.

11. The inhibitors of claim 10 characterized in that the linkers are cleavable 15 by coagulation proteases of the intrinsic coagulation pathway.

12. The inhibitors of claim 11 characterized by linkers cleavable by FXIla.

13. A pharmaceutical comprising an inhibitor as claimed in claims 1 through 20 12 or comprising an infestin or a fragment thereof.

14. An inhibitor as claimed in claims 1 through 12 and/or an Infestin or a fragment thereof for use as a medicament. 25

15. The use of an inhibitor as claimed in claims 1 through 12 and/or Infestin or fragments thereof for the preparation of a medicament for the treatment or prophylaxis of a condition or disorder related to arterial thrombus formation.

16. The use of an inhibitor as claimed in claims I through 12 and/or Infestin 30 or fragments thereof for the preparation of a medicament for the treatment or prophylaxis of arterial thrombosis, stroke, myocardial infarction, inflammation, complement activation, fibrinolysis, angiogenesis and/or diseases linked to pathological kinin formation such as hypotonic shock, edema including hereditary angioedema, bacterial infections, arthritis, pancreatitis, or articular gout, Disseminated Intravasal Coagulation (DIC) and sepsis. 5

17. Method to increase the efficacy of an inhibitor as claimed in claims I through 12 and/or Infestin or fragments thereof, preferably infestin 34 or Infestin-4 in the treatment of a condition or disorder related to arterial thrombus formation, wherein said inhibitors are linked to half-life enhancing polypeptides 10

18. Method to produce an inhibitor of claim 1 through claim 12 and/or Infestin or fragments thereof via recombinant means in eukaryotic cells, bacteria, yeasts, plant or insect cells or in transgenic animals. 15