Download PDF
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
1   Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany
,
Matthias J. E. Arlt
1   Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany
,
Markus M. Mueller
2   Max Planck Institut für Biochemie, AG Bioorganische Chemie, Martinsried, Germany
,
Stefan Sperl
3   Wilex AG, München, Germany
,
Axel Stemberger
1   Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany
,
Jörg Stürzebecher
4   Zentrum für Vaskuläre Biologie und Medizin, Klinikum der Universität Jena, Erfurt, Germany
,
Ali Amirkhosravi
5   Florida Hospital Cancer Institute, Orlando, Florida, USA
,
Luis Moroder
2   Max Planck Institut für Biochemie, AG Bioorganische Chemie, Martinsried, Germany
,
Achim Krüger
1   Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, München, Germany
› Author Affiliations Financial support: This work was supported by the German Research Council, SFB 469, project B13 (to AK) and grant LSHC-CT-2003-503297 “Cancerdegradome” from the European Union Framework Programme 6 (to AK).
Further Information

Publication History

Received: 13 April 2005

Accepted after revision: 01 August 2005

Publication Date:
14 December 2017 (online)

Also available at
    Permissions and Reprints

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
 

  • References

  • 1 Francis JL, Biggerstaff J, Amirkhosravi A. Hemostasis and malignancy. Semin Thromb Hemost 1998; 24: 93-109.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 3 Hoffman R, Haim N, Brenner B. Cancer and thrombosis revisited. Blood Rev 2001; 15: 61-7.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 6 De Cicco M. The prothrombotic state in cancer: pathogenic mechanisms. Crit Rev Oncol Hematol 2004; 50: 187-96.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 11 Sampson MT, Kakkar AK. Coagulation proteases and human cancer. Biochem Soc Trans 2002; 30: 201-7.
    CrossrefPubMedSearch in Google Scholar
  • 12 Hejna M, Raderer M, Zielinski CC. Inhibition of metastases by anticoagulants. J Natl Cancer Inst 1999; 91: 22-36.
    CrossrefPubMedSearch in Google Scholar
  • 13 Francis JL, Amirkhosravi A. Effect of antihemostatic agents on experimental tumor dissemination. Semin Thromb Hemost 2002; 28: 29-38.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 17 Amirkhosravi M, Francis JL. Coagulation activation by MC28 fibrosarcoma cells facilitates lung tumor formation. Thromb Haemost 1995; 73: 59-65.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 24 Krüger A, Soeltl R, Sopov I. et al. Hydroxamatetype matrix metalloproteinase inhibitor batimastat promotes liver metastasis. Cancer Res 2001; 61: 1272-5.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 26 Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 2002; 295: 2387-92.
    CrossrefPubMedSearch in Google Scholar
  • 27 Walsh PN, Ahmad SS. Proteases in blood clotting. Essays Biochem 2002; 38: 95-111.
    CrossrefPubMedSearch in Google Scholar
  • 28 Drew AF, Kaufman AH, Kombrinck KW. et al. Ligneous conjunctivitis in plasminogen-deficient mice. Blood 1998; 91: 1616-24.
    PubMedSearch in Google Scholar
  • 29 Neurath H. Proteolytic enzymes, past and future. Proc Natl Acad Sci U S A 1999; 96: 10962-3.
    CrossrefPubMedSearch in Google Scholar
  • 30 Yoshida S, Shiosaka S. Plasticity-related serine proteases in the brain (review). Int J Mol Med 1999; 3: 405-9.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 36 Dixon M. The determination of enzyme inhibitor constants. Biochem J 1953; 55: 170-1.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 40 Deitcher SR. Clinical utility of subcutaneous hirudins. Am J Health Syst Pharm 2003; 60 (Suppl. 05) S27-31.
    PubMedSearch in Google Scholar
  • 41 Mann KG, Nesheim ME, Church WR. et al. Surface- dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
    PubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 43 Kaiser B. DX-9065a, a direct inhibitor of factor Xa. Cardiovasc Drug Rev 2003; 21: 91-104.
    PubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 45 Camerer E, Qazi AA, Duong DN. et al. Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood 2004; 104: 397-401.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 48 McCulloch P, George WD. Warfarin inhibition of metastasis: the role of anticoagulation. Br J Surg 1987; 74: 879-83.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 51 DeClerck YA, Imren S, Montgomery AM. et al. Proteases and protease inhibitors in tumor progression. Adv Exp Med Biol 1997; 425: 89-97.
    PubMedSearch in Google Scholar