Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thr

1. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Jeanine M. Walenga, Walter P. Jeske, Margaret Prechel, Debra Hoppensteadt, Amanda Drenth, Jessica Swank, Meredith McDonald, Luke Sheen, Omer Iqbal, Brian Neville Presented at the XXIst Congress of International Society on Thrombosis and Haemostasis (ISTH) 2007 Meeting, July 6-12th in Geneva, Switzerland.

2. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Approximately 1�5% of patients receiving heparins (unfractionated heparin [UFH] and the low molecular weight heparins [LWMHs]) develop heparin-induced thrombocytopenia (HIT), which is associated with a substantially increased risk of thrombosis HIT is caused by the production of antibodies to a complex of heparin and platelet factor 4 (PF4), and subsequent cross-reaction of these HIT antibodies with heparin�PF4

3. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Current guidelines recommend the use of a direct thrombin inhibitor (DTI) in patients with HIT DTIs administered parenterally are associated with a high bleeding risk, as well as other drug-specific limitations

4. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Rivaroxaban is a novel, oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders Rivaroxaban may be suitable for the prevention and treatment of thrombosis in patients with HIT, because it is structurally unrelated to UFH, LMWHs and the heparin-based indirect FXa inhibitor fondaparinux, and, therefore, would not be expected to generate or cross-react with HIT antibodies

5. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia To evaluate, in vitro, the potential of rivaroxaban as an anticoagulant for the management of patients with HIT

6. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Several drugs were compared to rivaroxaban in the treatment of HIT: UFH the LMWH, enoxaparin fondaparinux the DTI, argatroban saline, as a control

7. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Serum was collected from 89 patients with HIT Three functional assays of platelet activation or aggregation were used to determine any cross-reaction between rivaroxaban, or other drugs, and HIT antibodies 14C-serotonin release assay (in washed platelets) Heparin-induced platelet aggregation assay (in platelet-rich plasma; platelet activation defined as >20% aggregation) Flow cytometric analysis of platelet activation (platelet microparticle formation, platelet aggregation, and platelet P-selectin expression [% positive platelets and mean fluorescence intensity]) in whole blood

8. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Several drugs were compared to rivaroxaban in the treatment of HIT: UFH The LMWH, enoxaparin Fondaparinux The DTI, argatroban Saline, as a control Rivaroxaban was tested in a total of 152 different HIT antibody/donor platelet combinations using the three assays

9. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia PF4 release assay: The release of PF4 from platelets was measured by ELISA Whole blood or platelet-rich plasma was incubated at 37�C for 30 minutes with stirring The assay was repeated after activation of platelets by tissue factor PF4 binding assay: The activity of study drugs (anti-FXa activity of rivaroxaban and fondaparinux; anti-thrombin activity of heparins and argatroban) was measured before and after incubation with 10 �g/ml purified PF4 at 37�C for 30 minutes, with stirring

10. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia 14C-serotonin release assay: Rivaroxaban, fondaparinux and argatroban did not activate platelets in the presence of HIT antibodies i.e. no concentration-dependent increase in activity As expected, strong, concentration-dependent platelet activation was observed with therapeutic concentrations of UFH and enoxaparin The absence of platelet activation at supra-therapeutic concentrations is typical of a heparin-dependent, platelet antibody activation response

11. Effect of rivaroxaban (n=28), fondaparinux (n=10), argatroban (n=10), enoxaparin (n=8) and unfractionated heparin (n=8) on platelet activation in the presence of heparin-induced thrombocytopenia antibodies, measured by the 14C-serotonin release assay. Units of concentration are ug/ml for rivaroxaban, fondaparinux and argatroban, and U/ml for enoxaparin and unfractionated heparin.Effect of rivaroxaban (n=28), fondaparinux (n=10), argatroban (n=10), enoxaparin (n=8) and unfractionated heparin (n=8) on platelet activation in the presence of heparin-induced thrombocytopenia antibodies, measured by the 14C-serotonin release assay. Units of concentration are ug/ml for rivaroxaban, fondaparinux and argatroban, and U/ml for enoxaparin and unfractionated heparin.

12. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Heparin-induced platelet aggregation assay: Rivaroxaban did not activate platelets in the presence of HIT antibodies Fondaparinux caused activation of platelets in only one of the 18 sera samples tested UFH and enoxaparin strongly induced platelet activation in 100% and 70% of sera samples, respectively

13. Effect of rivaroxaban, fondaparinux, enoxaparin and unfractionated heparin (UFH) on platelet activation (defined as >20% aggregation) in the presence of heparin-induced thrombocytopenia antibodies (n=18), as measured by the heparin-induced platelet aggregation assay. Effect of rivaroxaban, fondaparinux, enoxaparin and unfractionated heparin (UFH) on platelet activation (defined as >20% aggregation) in the presence of heparin-induced thrombocytopenia antibodies (n=18), as measured by the heparin-induced platelet aggregation assay.

14. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Flow cytometric analysis of platelet activation: Rivaroxaban, fondaparinux and argatroban did not activate platelets in the presence of HIT antibodies (i.e. no concentration-dependent increase in parameters of platelet activation; data not shown) UFH and enoxaparin induced strong, concentration-dependent platelet activation in the presence of HIT antibodies (data not shown) PF4 release assay: Rivaroxaban and fondaparinux did not activate platelets, as measured by PF4 release UFH and enoxaparin caused strong platelet activation

15. Effect of rivaroxaban, fondaparinux, enoxaparin, and unfractionated heparin (UFH) on platelet factor 4 (PF4) release in whole blood (n=5 per drug). A similar pattern was observed in platelet-rich plasma with tissue factor activation.Effect of rivaroxaban, fondaparinux, enoxaparin, and unfractionated heparin (UFH) on platelet factor 4 (PF4) release in whole blood (n=5 per drug). A similar pattern was observed in platelet-rich plasma with tissue factor activation.

16. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia PF4 binding assay: The anti-FXa activity of rivaroxaban and fondaparinux was unchanged after incubation with PF4, as was the anti-thrombin activity of argatroban The anti-thrombin activity of UFH and enoxaparin was decreased by 64% and 43%, respectively

17. Effect of platelet factor 4 (PF4) on the anti-factor Xa (anti-Xa) activity of rivaroxaban and fondaparinux and the anti-thrombin (anti-IIa) activity of argatroban, enoxaparin, and unfractionated heparin (UFH).Effect of platelet factor 4 (PF4) on the anti-factor Xa (anti-Xa) activity of rivaroxaban and fondaparinux and the anti-thrombin (anti-IIa) activity of argatroban, enoxaparin, and unfractionated heparin (UFH).

18. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Unlike UFH and enoxaparin, Rivaroxaban Did not cross-react with HIT antibodies Did not interact with PF4 Did not mobilize PF4 from platelets Rivaroxaban may be considered an alternative anticoagulant for the management of patients with HIT

19. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Rivaroxaban, if used as the initial anticoagulant in place of heparin, may not initiate HIT antibody production Rivaroxaban, administered orally, could be used for long-term secondary prevention after an acute episode of HIT

20. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Further clinical investigation is required to confirm the potential of rivaroxaban as an alternative anticoagulant for the management of patients with HIT Rivaroxaban may offer certain advantages over currently approved DTIs in patients with HIT, due to its Oral route of administration Wide therapeutic window And potentially improved bleeding profile