Effective inhibition of experimental metastasis and prolongation of survival in mice by a potent factor Xa-specific synthetic serine protease inhibitor with weak anticoagulant activity

Ingo J. Banke; Matthias J. E. Arlt; Markus M. Mueller; Stefan Sperl; Axel Stemberger; Jörg Stürzebecher; Ali Amirkhosravi; Luis Moroder; Achim Krüger

doi:10.1160/TH05-04-0249

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 94(05): 1084-1093
DOI: 10.1160/TH05-04-0249

Cellular Proteolysis and Oncology

Schattauer GmbH

Effective inhibition of experimental metastasis and prolongation of survival in mice by a potent factor Xa-specific synthetic serine protease inhibitor with weak anticoagulant activity

Ingo J. Banke

¹Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany

,

Matthias J. E. Arlt

¹Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany

,

Markus M. Mueller

²Max Planck Institut für Biochemie, AG Bioorganische Chemie, Martinsried, Germany

,

Stefan Sperl

³Wilex AG, München, Germany

,

Axel Stemberger

¹Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany

,

Jörg Stürzebecher

⁴Zentrum für Vaskuläre Biologie und Medizin, Klinikum der Universität Jena, Erfurt, Germany

,

Ali Amirkhosravi

⁵Florida Hospital Cancer Institute, Orlando, Florida, USA

,

Luis Moroder

²Max Planck Institut für Biochemie, AG Bioorganische Chemie, Martinsried, Germany

,

Achim Krüger

¹Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany

› Author Affiliations

Abstract

Summary

Clinical and experimental evidence suggests that the blood coagulation system is involved in the dissemination of malignant tumors. Consequently, anticoagulant agents have been tested as metastasis suppressors in experimental models. Recently, we have found a close correlation between factor Xa (FXa)-specificity of a series of synthetic serine protease inhibitors and their anti-metastatic potential in a murineT-cell lymphoma metastasis model. Interference of such inhibitors with blood-coagulation may represent a major experimental and clinical obstacle. Here, we test anti-metastatic effects of a recently developed, highly specific 3-amidinophenylalanine-type FXa inhibitor, WX-FX4, with weaker anticoagulant activity when compared to well-established FXa inhibitors, such as DX-9065a, as measured by the activated partial thromboplastin time, prothrombin time, prothrombinase complex activity, and coagulation time. Treatment of mice with WX-FX4 (1.5 mg/kg twice daily) led to significant reduction of experimental liver metastasis of a syngeneic T-cell lymphoma in DBA/2 mice (> 90%), and of experimental lung metastasis of a human fibrosarcoma in CD1 nu/nu mice (> 60%). Due to its relatively low anticoagulant activity, daily treatment over 100 days was possible, leading to significant survival benefits without inducing bleeding anomalities. FXa-inhibitors with highly efficient anti-metastatic potential without coagulationrelated side effects may represent important new tools as anticancer agents.

Keywords

Coagulation Factor Xa inhibitor - metastasis - lymphoma - fibrosarcoma

Full Text

References

References
1 Francis JL, Biggerstaff J, Amirkhosravi A. Hemostasis and malignancy. Semin Thromb Hemost 1998; 24: 93-109.
2 Zacharski LR. Malignancy as a solid-phase coagulopathy: implications for the etiology, pathogenesis, and treatment of cancer. Semin Thromb Hemost 2003; 29: 239-46.
3 Hoffman R, Haim N, Brenner B. Cancer and thrombosis revisited. Blood Rev 2001; 15: 61-7.
4 Bura A, Cailleux N, Bienvenu B. et al. Incidence and prognosis of cancer associated with bilateral venous thrombosis: a prospective study of 103 patients. J Thromb Haemost 2004; 2: 441-4.
5 Sallah S, Wan JY, Nguyen NP. et al. Disseminated intravascular coagulation in solid tumors: clinical and pathologic study. Thromb Haemost 2001; 86: 828-33.
6 De Cicco M. The prothrombotic state in cancer: pathogenic mechanisms. Crit Rev Oncol Hematol 2004; 50: 187-96.
7 Wicki J, Perrier A, Perneger TV. et al. Predicting adverse outcome in patients with acute pulmonary embolism: a risk score. Thromb Haemost 2000; 84: 548-52.
8 Hembrough TA, Swartz GM, Papathanassiu A. et al. Tissue factor/factor VIIa inhibitors block angiogenesis and tumor growth through a nonhemostatic mechanism. Cancer Res 2003; 63: 2997-3000.
9 Palumbo JS, Kombrinck KW, Drew AF. et al. Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells. Blood 2000; 96: 3302-9.
10 Hu L, Lee M, Campbell W. et al. Role of endogenous thrombin in tumor implantation, seeding, and spontaneous metastasis. Blood 2004; 104: 2746-51.
11 Sampson MT, Kakkar AK. Coagulation proteases and human cancer. Biochem Soc Trans 2002; 30: 201-7.
12 Hejna M, Raderer M, Zielinski CC. Inhibition of metastases by anticoagulants. J Natl Cancer Inst 1999; 91: 22-36.
13 Francis JL, Amirkhosravi A. Effect of antihemostatic agents on experimental tumor dissemination. Semin Thromb Hemost 2002; 28: 29-38.
14 Ornstein DL, Zacharski LR. Activity of heparins in experimental models of malignancy: Possible explanations for anticancer effects in humans. Clin Hemorheol Microcirc 2002; 26: 33-40.
15 Amirkhosravi A, Mousa SA, Amaya M. et al. Antimetastatic effect of tinzaparin, a low-molecular-weight heparin. J Thromb Haemost 2003; 1: 1972-6.
16 Francis JL, Carty N, Amirkhosravi M. et al. The effect of Warfarin and factor VII on tissue procoagulant activity and pulmonary seeding. Br J Cancer 1992; 65: 329-34.
17 Amirkhosravi M, Francis JL. Coagulation activation by MC28 fibrosarcoma cells facilitates lung tumor formation. Thromb Haemost 1995; 73: 59-65.
18 Leadley Jr. RJ, Chi L, Porcari AR. Non-hemostatic activity of coagulation factor Xa: potential implications for various diseases. Curr Opin Pharmacol 2001; 1: 169-75.
19 Jiang X, Bailly MA, Panetti TS. et al. Formation of tissue factor-factor VIIa-factor Xa complex promotes cellular signaling and migration of human breast cancer cells. J Thromb Haemost 2004; 2: 93-101.
20 Tuszynski GP, Gasic TB, Gasic GJ. Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation. J Biol Chem 1987; 262: 9718-23.
21 Donnelly KM, Bromberg ME, Milstone A. et al. Ancylostoma caninum anticoagulant peptide blocks metastasis in vivo and inhibits factor Xa binding to melanoma cells in vitro . Thromb Haemost 1998; 79: 1041-7.
22 Banke IJ, Arlt MJ, Pennington C. et al. Increase of anti-metastatic efficacy by selectivity- but not affinityoptimization of synthetic serine protease inhibitors. Biol Chem 2003; 384: 1515-25.
23 Gould WR, Leadley RJ. Recent advances in the discovery and development of direct coagulation factor Xa inhibitors. Curr Pharm Des 2003; 9: 2337-47.
24 Krüger A, Soeltl R, Sopov I. et al. Hydroxamatetype matrix metalloproteinase inhibitor batimastat promotes liver metastasis. Cancer Res 2001; 61: 1272-5.
25 Arlt M, Kopitz C, Pennington C. et al. Increase in gelatinase-specificity of matrix metalloproteinase inhibitors correlates with antimetastatic efficacy in a T-cell lymphoma model. Cancer Res 2002; 62: 5543-50.
26 Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 2002; 295: 2387-92.
27 Walsh PN, Ahmad SS. Proteases in blood clotting. Essays Biochem 2002; 38: 95-111.
28 Drew AF, Kaufman AH, Kombrinck KW. et al. Ligneous conjunctivitis in plasminogen-deficient mice. Blood 1998; 91: 1616-24.
29 Neurath H. Proteolytic enzymes, past and future. Proc Natl Acad Sci U S A 1999; 96: 10962-3.
30 Yoshida S, Shiosaka S. Plasticity-related serine proteases in the brain (review). Int J Mol Med 1999; 3: 405-9.
31 Mueller MM, Sperl S, Stürzebecher J. et al. (R)- 3-Amidinophenylalanine-derived inhibitors of factor Xa with a novel active-site binding mode. Biol Chem 2002; 383: 1185-91.
32 Krüger A, Umansky V, Rocha M. et al. Pattern and load of spontaneous liver metastasis dependent on host immune status studied with a lacZ transduced lymphoma. Blood 1994; 84: 3166-74.
33 Krüger A, Schirrmacher V, Khokha R. The bacterial lacZ gene: an important tool for metastasis research and evaluation of new cancer therapies. Cancer Metastasis Rev 1998; 17: 285-94.
34 Nagahara T, Yokoyama Y, Inamura K. et al. Dibasic (amidinoaryl)propanoic acid derivatives as novel blood coagulation factor Xa inhibitors. J Med Chem 1994; 37: 1200-7.
35 Stürzebecher J, Prasa D, Hauptmann J. et al. Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine. J Med Chem 1997; 40: 3091-9.
36 Dixon M. The determination of enzyme inhibitor constants. Biochem J 1953; 55: 170-1.
37 Hauptmann J, Steinmetzer T, Vieweg H. et al. Influence of structural variations in peptidomimetic 4– amidinophenylalanine-derived thrombin inhibitors on plasma clearance and biliary excretion in rats. Pharm Res 2002; 19: 1027-33.
38 Schweinitz A, Steinmetzer T, Banke IJ. et al. Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents. J Biol Chem 2004; 279: 33613-22.
39 Sinha U, Lin PH, Edwards ST. et al. Inhibition of purified factor Xa amidolytic activity may not be predictive of inhibition of in vivo thrombosis: implications for identification of therapeutically active inhibitors. Arterioscler Thromb Vasc Biol 2003; 23: 1098-104.
40 Deitcher SR. Clinical utility of subcutaneous hirudins. Am J Health Syst Pharm 2003; 60 (Suppl. 05) S27-31.
41 Mann KG, Nesheim ME, Church WR. et al. Surface- dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
42 Rezaie AR. DX-9065a inhibition of factor Xa and the prothrombinase complex: mechanism of inhibition and comparison with therapeutic heparins. Thromb Haemost 2003; 89: 112-21.
43 Kaiser B. DX-9065a, a direct inhibitor of factor Xa. Cardiovasc Drug Rev 2003; 21: 91-104.
44 Amirkhosravi A, Mousa SA, Amaya M. et al. Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454. Thromb Haemost 2003; 90: 549-54.
45 Camerer E, Qazi AA, Duong DN. et al. Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood 2004; 104: 397-401.
46 Palumbo JS, Talmage KE, Massari JV. et al. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood 2005; 105: 178-85.
47 Camerer E, Huang W, Coughlin SR. Tissue factorand factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Natl Acad Sci U S A 2000; 97: 5255-60.
48 McCulloch P, George WD. Warfarin inhibition of metastasis: the role of anticoagulation. Br J Surg 1987; 74: 879-83.
49 Koop S, Schmidt EE, MacDonald IC. et al. Independence of metastatic ability and extravasation: metastatic ras- transformed and control fibroblasts extravasate equally well. Proc Natl Acad Sci U S A 1996; 93: 11080-4.
50 Naumov GN, Wilson SM, MacDonald IC. et al. Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis. J Cell Sci 1999; 112: 1835-42.
51 DeClerck YA, Imren S, Montgomery AM. et al. Proteases and protease inhibitors in tumor progression. Adv Exp Med Biol 1997; 425: 89-97.