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Thromb Haemost 2007; 97(05): 847-855
DOI: 10.1160/TH06-12-0732
Animal Models
Schattauer GmbH

A novel P2Y12 adenosine diphosphate receptor antagonist that inhibits platelet aggregation and thrombus formation in rat and dog models

Yi-Xin Wang
1   Berlex Biosciences, Richmond, California, USA
,
Jon Vincelette
1   Berlex Biosciences, Richmond, California, USA
,
Valdeci da Cunha
1   Berlex Biosciences, Richmond, California, USA
,
Baby Martin-McNulty
1   Berlex Biosciences, Richmond, California, USA
,
Cornell Mallari
1   Berlex Biosciences, Richmond, California, USA
,
Richard M. Fitch
1   Berlex Biosciences, Richmond, California, USA
,
Serene Alexander
1   Berlex Biosciences, Richmond, California, USA
,
Imadul Islam
1   Berlex Biosciences, Richmond, California, USA
,
Brad O. Buckman
1   Berlex Biosciences, Richmond, California, USA
,
Shendong Yuan
1   Berlex Biosciences, Richmond, California, USA
,
Joseph M. Post
1   Berlex Biosciences, Richmond, California, USA
,
Babu Subramanyam
1   Berlex Biosciences, Richmond, California, USA
,
Ronald Vergona
1   Berlex Biosciences, Richmond, California, USA
,
Mark E. Sullivan
1   Berlex Biosciences, Richmond, California, USA
,
William P. Dole
1   Berlex Biosciences, Richmond, California, USA
,
John Morser
1   Berlex Biosciences, Richmond, California, USA
,
Judi Bryant
1   Berlex Biosciences, Richmond, California, USA
› Author Affiliations
Further Information

Publication History

Received 22 December 2006

Accepted after revision 01 March 2007

Publication Date:
24 November 2017 (online)

Also available at
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Summary

Irreversible platelet inhibitors, such as aspirin and clopidogrel, have limited anti-thrombotic efficacy in the clinic due to their bleeding risk. We have developed an orally active reversible P2Y12 receptor antagonist, BX 667.The aim of this study was to determine if the reversible antagonist BX 667 had a greater therapeutic index than the irreversible P2Y12 receptor antagonist clopidogrel. Since BX 667 is rapidly converted to its active metabolite BX 048 in rats,we first injected BX 048 intravenously (iv) in a rat arterial venous (A-V) shunt model of thrombosis.BX 048 dose- and concentration-dependently attenuated thrombosis. When administered orally, BX 667 and clopidogrel had similar efficacy, but BX 667 caused less bleeding than clopidogrel. In a rat model of a platelet-rich thrombus induced by vessel injury with FeCl2, both BX 667 and clopidogrel exhibited higher levels of thrombus inhibition after oral administration compared to their potency in the A-V shunt model.Again, BX 667 caused less bleeding than clopidogrel. In a dog cyclic flow model, iv injection of either BX 667 or clopidogrel dose-dependently reduced thrombus formation with lower bleeding for BX 667 than clopidogrel. Inhibition of thrombosis was highly correlated with inhibition of ADP-induced platelet aggregation in these animal models. In dogs pre-treated with aspirin, BX 667 maintained its wider therapeutic index, measured by inhibition of platelet aggregation over bleeding, compared to the aspirin-clopidogrel combination.These data demonstrate that the reversible P2Y12 receptor antagonist, BX 667, has a wider therapeutic index than clopidogrel in experimental models of thrombosis.

Keywords

ADP - platelet aggregation - thrombosis - rat - dog - cyclic flow variation
 

  • References

  • 1 Roth GJ, Stanford N, Majerus PW. Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 1975; 72: 3073-3076.
    CrossrefPubMedSearch in Google Scholar
  • 2 Antiplatelet-Trialists'-Collaboration-I. Collaborative overview of randomised trials of antiplatelet therapy-- I: Prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. Br Med J 1994; 308: 81-106.
    CrossrefPubMedSearch in Google Scholar
  • 3 Antiplatelet-Trialists'-Collaboration-II. Collaborative overview of randomised trials of antiplatelet therapy-- II: Maintenance of vascular graft or arterial patency by antiplatelet therapy. Br Med J 1994; 308: 159-168.
    CrossrefPubMedSearch in Google Scholar
  • 4 Antiplatelet-Trialists'-Collaboration-III. Collaborative overview of randomised trials of antiplatelet therapy-- III: Reduction in venous thrombosis and pulmonary embolism by antiplatelet prophylaxis among surgical and medical patients. Br Med J 1994; 308: 235-246.
    CrossrefPubMedSearch in Google Scholar
  • 5 Savi P, Herbert JM, Pflieger AM. et al. Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel. Biochem Pharmacol 1992; 44: 527-532.
    CrossrefPubMedSearch in Google Scholar
  • 6 Pereillo JM, Maftouh M, Andrieu A. et al. Structure and stereochemistry of the active metabolite of clopidogrel. Drug Metab Dispos 2002; 30: 1288-1295.
    CrossrefPubMedSearch in Google Scholar
  • 7 Savi P, Pereillo JM, Uzabiaga MF. et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost 2000; 84: 891-896.
    Thieme ConnectPubMedSearch in Google Scholar
  • 8 Savi P, Herbert JM. ADP receptors on platelets and ADP-selective antiaggregating agents. Med Res Rev 1996; 16: 159-179.
    CrossrefPubMedSearch in Google Scholar
  • 9 Hollopeter G, Jantzen HM, Vincent D. et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001; 409: 202-207.
    CrossrefPubMedSearch in Google Scholar
  • 10 CAPRIE-Steering-Committee. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet 1996; 348: 1329-1339.
    CrossrefPubMedSearch in Google Scholar
  • 11 Harker LA, Boissel JP, Pilgrim AJ. et al. Comparative safety and tolerability of clopidogrel and aspirin: results from CAPRIE. CAPRIE Steering Committee and Investigators. Clopidogrel versus aspirin in patients at risk of ischaemic events. Drug Saf 1999; 21: 325-335.
    CrossrefPubMedSearch in Google Scholar
  • 12 CURE-Trial. The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 2001; 2001 345: 494-502.
    CrossrefPubMedSearch in Google Scholar
  • 13 Thebault JJ, Kieffer G, Lowe GD. et al. Repeateddose pharmacodynamics of clopidogrel in healthy subjects. Semin Thromb Hemost 1999; 25 (Suppl. 02) (Suppl) 9-14.
    PubMedSearch in Google Scholar
  • 14 Caplain H, Cariou R. Long-term activity of clopidogrel: a three-month appraisal in healthy volunteers. Semin Thromb Hemost 1999; 25 (Suppl. 02) (Suppl) 21-24.
    PubMedSearch in Google Scholar
  • 15 Caplain H, D'Honneur G, Cariou R. Prolonged heparin administration during clopidogrel treatment in healthy subjects. Semin Thromb Hemost 1999; 25 (Suppl. 02) (Suppl 2) 61-64.
    PubMedSearch in Google Scholar
  • 16 Caplain H, Donat F, Gaud C. et al. Pharmacokinetics of clopidogrel. Semin Thromb Hemost 1999; 25 (Suppl. 02) (Suppl) 25-28.
    PubMedSearch in Google Scholar
  • 17 Caplain H, Thebault JJ, Necciari J. Clopidogrel does not affect the pharmacokinetics of theophylline. Semin Thromb Hemost 1999; 25 (Suppl. 02) (Suppl) 65-68.
    Thieme ConnectPubMedSearch in Google Scholar
  • 18 Steinhubl SR, Berger PB, Brennan DM. et al. Optimal timing for the initiation of pre-treatment with 300 mg clopidogrel before percutaneous coronary intervention. J Am Coll Cardiol 2006; 47: 939-943.
    CrossrefPubMedSearch in Google Scholar
  • 19 Steinhubl SR, Berger PB, Mann JT 3rd. et al. Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial. J Am Med Assoc 2002; 288: 2411-2420.
    CrossrefPubMedSearch in Google Scholar
  • 20 Bennett CL, Connors JM, Carwile JM. et al. Thrombotic thrombocytopenic purpura associated with clopidogrel. N Engl J Med 2000; 342: 1773-1777.
    CrossrefPubMedSearch in Google Scholar
  • 21 Lau WC, Gurbel PA. Antiplatelet drug resistance and drug-drug interactions: Role of cytochrome P450 3A4. Pharm Res 2006; 23: 2691-2708.
    CrossrefPubMedSearch in Google Scholar
  • 22 Neubauer H, Mugge A. Thienopyridines and statins: assessing a potential drug-drug interaction. Curr Pharm Des 2006; 12: 1271-1280.
    CrossrefPubMedSearch in Google Scholar
  • 23 Cattaneo M. Aspirin and clopidogrel: Efficacy, safety, and the issue of drug resistance. 2004; 1980-7.
    PubMedSearch in Google Scholar
  • 24 Cattaneo M, Gachet C. ADP receptors and clinical bleeding disorders. Arterioscler Thromb Vasc Biol 1999; 19: 2281-2285.
    CrossrefPubMedSearch in Google Scholar
  • 25 Nguyen TA, Diodati JG, Pharand C. Resistance to clopidogrel: a review of the evidence. J Am Coll Cardiol 2005; 45: 1157-1164.
    CrossrefPubMedSearch in Google Scholar
  • 26 Wang TH, Bhatt DL, Topol EJ. Aspirin and clopidogrel resistance: an emerging clinical entity. Eur Heart J 2006; 27: 647-654.
    CrossrefPubMedSearch in Google Scholar
  • 27 Falati S, Gross P, Merrill-Skoloff G. et al. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med 2002; 08: 1175-1181.
    CrossrefPubMedSearch in Google Scholar
  • 28 Falati S, Gross PL, Merrill-Skoloff G. et al. In vivo models of platelet function and thrombosis: study of real-time thrombus formation. Methods Mol Biol 2004; 272: 187-197.
    PubMedSearch in Google Scholar
  • 29 Wang YX, Dong N, Wu C. et al. Lipopolysaccharide attenuates thrombolysis in batroxobin-induced lung vasculature fibrin deposition but not in ferrous chloride- induced carotid artery thrombus in rats: role of endogenous PAI-1. Thromb Res 2003; 111: 381-387.
    CrossrefPubMedSearch in Google Scholar
  • 30 Folts JD, Crowell EB Jr, Rowe GG. Platelet aggregation in partially obstructed vessels and its elimination with aspirin. Circulation 1976; 54: 365-370.
    CrossrefPubMedSearch in Google Scholar
  • 31 Eidt JF, Ashton J, Golino P. et al. Thromboxane A2 and serotonin mediate coronary blood flow reductions in unsedated dogs. Am J Physiol 1989; 257: H873-882.
    PubMedSearch in Google Scholar
  • 32 Eidt JF, Ashton J, Golino P. et al. Treadmill exercise promotes cyclic alterations in coronary blood flow in dogs with coronary artery stenoses and endothelial injury. J Clin Invest 1989; 84: 517-527.
    CrossrefPubMedSearch in Google Scholar
  • 33 Eidt JF, Allison P, Noble S. et al. Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury. J Clin Invest 1989; 84: 18-27.
    CrossrefPubMedSearch in Google Scholar
  • 34 Eidt JF, Allison P, Noble S. et al. Thrombin is an important mediator of cyclic coronary blood flow variations due to platelet aggregation in stenosed canine coronary arteries. Trans Assoc Am Physicians 1988; 101: 125-136.
    PubMedSearch in Google Scholar
  • 35 Willerson JT, Yao SK, McNatt J. et al. Frequency and severity of cyclic flow alternations and platelet aggregation predict the severity of neointimal proliferation following experimental coronary stenosis and endothelial injury. Proc Natl Acad Sci USA 1991; 88: 10624-10628.
    CrossrefPubMedSearch in Google Scholar
  • 36 Huang J, Driscoll EM, Gonzales ML. et al. Prevention of arterial thrombosis by intravenously administered platelet P2T receptor antagonist AR-C69931MX in a canine model. J Pharmacol Exp Ther 2000; 295: 492-499.
    PubMedSearch in Google Scholar
  • 37 Robertson MJ, Humphries RG, Tomlinson W. et al. The effects of FPL 67085 on thrombosis and haemostasis in the anaesthetized dog: Comparison with aspirin and the GPIIb/IIIa antagonists, Ro 449883 and GR 144053. Br J Pharmacol 1994; 113: 64P
    PubMedSearch in Google Scholar
  • 38 van Giezen JJ, Humphries RG. Preclinical and clinical studies with selective reversible direct P2Y12 antagonists. Semin Thromb Hemost 2005; 31: 195-204.
    Thieme ConnectPubMedSearch in Google Scholar
  • 39 Wang K, Zhou X, Zhou Z. et al. Blockade of the platelet P2Y12 receptor by AR-C69931MX sustains coronary artery recanalization and improves the myocardial tissue perfusion in a canine thrombosis model. Arterioscler Thromb Vasc Biol 2003 2003; 23: 357-362.
    PubMedSearch in Google Scholar
  • 40 Storey RF, Wilcox RG, Heptinstall S. Comparison of the pharmacodynamic effects of the platelet ADP receptor antagonists clopidogrel and AR-C69931MX in patients with ischaemic heart disease. Platelets 2002; 13: 407-413.
    CrossrefPubMedSearch in Google Scholar
  • 41 Wyld PJ, Gardner JJ, Wilkinson D. et al. Dose-response relationship and elimination characteristics of the short-acting P2T-purinoceptor antagonist FPL 67085 in man. Blood 1994; 84 (Suppl. 01) (Suppl) 475a
    PubMedSearch in Google Scholar
  • 42 Greenbaum AB, Grines CL, Bittl JA. et al. Initial experience with an intravenous P2Y12 platelet receptor antagonist in patients undergoing percutaneous coronary intervention: results from a 2-part, phase II, multicenter, randomized, placebo- and active-controlled trial. Am Heart J 2006; 151: 689.e1-e10.
    CrossrefPubMedSearch in Google Scholar
  • 43 Ono M, Nawa K, Marumoto Y. Antithrombotic effects of recombinant human soluble thrombomodulin in a rat model of vascular shunt thrombosis. Thromb Haemost 1994; 72: 421-425.
    Thieme ConnectPubMedSearch in Google Scholar
  • 44 Berry CN, Girard D, Lochot S. et al. Antithrombotic actions of argatroban in rat models of venous, 'mixed' and arterial thrombosis, and its effects on the tail transection bleeding time. Br J Pharmacol 1994; 113: 1209-1214.
    CrossrefPubMedSearch in Google Scholar
  • 45 Dong N, Da Cunha V, Citkowicz A. et al. P-selectintargeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator alpha1. Thromb Haemost 2004; 92: 956-965.
    Thieme ConnectPubMedSearch in Google Scholar
  • 46 Copeland RA, Pompliano DL, Meek TD. Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov 2006; 05: 730-739.
    CrossrefPubMedSearch in Google Scholar