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Slide 1 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451
Slide 2 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations
Slide 3 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding?
Slide 4 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity
Slide 5 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity In vivo rat model - reversal of anticoagulation 5 Rapid normalisation of INR after r-antidote infusion Reversal of anticoagulation correlates to reduction of unbound fXa-inhibitor-fraction
Slide 6 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity In vivo rat model - reversal of anticoagulation 5 Rapid normalisation of INR after r-antidote infusion Reversal of anticoagulation correlates to reduction of unbound fXa-inhibitor-fraction Mouse model of blood loss (tail transection) ASS+Rivar.+r-antidote reduced blood loss by 85% After r-antidote rivaroxaban activity reduced by >80% 6 Treat-I: vehicle + vehicle + vehicle Treat-II: vehicle + rivaroxaban + vehicle Treat-III: ASA + vehicle + vehicle Treat-IV: ASA + rivaroxaban + vehicle Treat-V: ASA + rivaroxaban + r- Antidote
Slide 7 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity In vivo rat model - reversal of anticoagulation 5 Rapid normalisation of INR after r-antidote infusion Reversal of anticoagulation correlates to reduction of unbound fXa-inhibitor-fraction Mouse model of blood loss (tail transection) ASS+Rivar.+r-antidote reduced blood loss by 85% After r-antidote rivaroxaban activity reduced by >80% 6 Treat-I: vehicle + vehicle + vehicle Treat-II: vehicle + rivaroxaban + vehicle Treat-III: ASA + vehicle + vehicle Treat-IV: ASA + rivaroxaban + vehicle Treat-V: ASA + rivaroxaban + r- Antidote Rabbit model of blood loss (liver laceration model) Laceration of 2 liver lobes and blood loss collection over 15min after injection of vehicle or rivaroxaban and r-antidote 7 PT & aPTT increased 2.3x resp. 1.9x after rivaroxaban, blood loss 3.2x higher r-Antidote reduced blood loss by >85%, decreased anti-fXa activity by 98% Correlation of blood loss with reduction in anti-fXa-activity an free fXa-inhibiton
Slide 8 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity In vivo rat model - reversal of anticoagulation 5 Rapid normalisation of INR after r-antidote infusion Reversal of anticoagulation correlates to reduction of unbound fXa-inhibitor-fraction Mouse model of blood loss (tail transection) ASS+Rivar.+r-antidote reduced blood loss by 85% After r-antidote rivaroxaban activity reduced by >80% 6 Treat-I: vehicle + vehicle + vehicle Treat-II: vehicle + rivaroxaban + vehicle Treat-III: ASA + vehicle + vehicle Treat-IV: ASA + rivaroxaban + vehicle Treat-V: ASA + rivaroxaban + r- Antidote Rabbit model of blood loss (liver laceration model) Laceration of 2 liver lobes and blood loss collection over 15min after injection of vehicle or rivaroxaban and r-antidote 7 PT & aPTT increased 2.3x resp. 1.9x after rivaroxaban, blood loss 3.2x higher r-Antidote reduced blood loss by >85%, decreased anti-fXa activity by 98% Correlation of blood loss with reduction in anti-fXa-activity an free fXa-inhibiton Reversal of ATIII-dependent fXa inhibitors by r-antidote r-antidote mimicking binding-site of fXa for ATIII Enoxaparin (LMWH) & Fondaparinux (pentasaccharide) indirect fXa inhibitor (increase affinity of ATIII for fXa) 8 Fondaparinux Enoxaparin anti-fXa activitiy of enoxaparin & fondaparinux dose-dependently reversed with r-antidote
Slide 9 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity In vivo rat model - reversal of anticoagulation 5 Rapid normalisation of INR after r-antidote infusion Reversal of anticoagulation correlates to reduction of unbound fXa-inhibitor-fraction Mouse model of blood loss (tail transection) ASS+Rivar.+r-antidote reduced blood loss by 85% After r-antidote rivaroxaban activity reduced by >80% 6 Treat-I: vehicle + vehicle + vehicle Treat-II: vehicle + rivaroxaban + vehicle Treat-III: ASA + vehicle + vehicle Treat-IV: ASA + rivaroxaban + vehicle Treat-V: ASA + rivaroxaban + r- Antidote Rabbit model of blood loss (liver laceration model) Laceration of 2 liver lobes and blood loss collection over 15min after injection of vehicle or rivaroxaban and r-antidote 7 PT & aPTT increased 2.3x resp. 1.9x after rivaroxaban, blood loss 3.2x higher r-Antidote reduced blood loss by >85%, decreased anti-fXa activity by 98% Correlation of blood loss with reduction in anti-fXa-activity an free fXa-inhibiton Reversal of ATIII-dependent fXa inhibitors by r-antidote r-antidote mimicking binding-site of fXa for ATIII Enoxaparin (LMWH) & Fondaparinux (pentasaccharide) indirect fXa inhibitor (increase affinity of ATIII for fXa) 8 Fondaparinux Enoxaparin anti-fXa activitiy of enoxaparin & fondaparinux dose-dependently reversed with r-antidote Reversal of ATIII-dependent fXa inhibitors by r-antidote Animal model (rat tail transection) 9 Treat-I: vehicle Treat-II: Enoxaparin & vehicle Treat-III Enoxaparin & r-antidote 2mg/h Treat-IV: Enoxaparin & r-antidote 4mg/h Enoxaparin Treat-I: vehicle Treat-II: Fondaparinux & vehicle Treat-III Fondaparinux & r-antidote Treat-IV: fondaparinux & Protamine Normalisation of hemostasis after r-antidote administration Complete reversal of ATIII-dependent fXa inhibition
Slide 10 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity In vivo rat model - reversal of anticoagulation 5 Rapid normalisation of INR after r-antidote infusion Reversal of anticoagulation correlates to reduction of unbound fXa-inhibitor-fraction Mouse model of blood loss (tail transection) ASS+Rivar.+r-antidote reduced blood loss by 85% After r-antidote rivaroxaban activity reduced by >80% 6 Treat-I: vehicle + vehicle + vehicle Treat-II: vehicle + rivaroxaban + vehicle Treat-III: ASA + vehicle + vehicle Treat-IV: ASA + rivaroxaban + vehicle Treat-V: ASA + rivaroxaban + r- Antidote Rabbit model of blood loss (liver laceration model) Laceration of 2 liver lobes and blood loss collection over 15min after injection of vehicle or rivaroxaban and r-antidote 7 PT & aPTT increased 2.3x resp. 1.9x after rivaroxaban, blood loss 3.2x higher r-Antidote reduced blood loss by >85%, decreased anti-fXa activity by 98% Correlation of blood loss with reduction in anti-fXa-activity an free fXa-inhibiton Reversal of ATIII-dependent fXa inhibitors by r-antidote r-antidote mimicking binding-site of fXa for ATIII Enoxaparin (LMWH) & Fondaparinux (pentasaccharide) indirect fXa inhibitor (increase affinity of ATIII for fXa) 8 Fondaparinux Enoxaparin anti-fXa activitiy of enoxaparin & fondaparinux dose-dependently reversed with r-antidote Reversal of ATIII-dependent fXa inhibitors by r-antidote Animal model (rat tail transection) 9 Treat-I: vehicle Treat-II: Enoxaparin & vehicle Treat-III Enoxaparin & r-antidote 2mg/h Treat-IV: Enoxaparin & r-antidote 4mg/h Enoxaparin Treat-I: vehicle Treat-II: Fondaparinux & vehicle Treat-III Fondaparinux & r-antidote Treat-IV: fondaparinux & Protamine Normalisation of hemostasis after r-antidote administration Complete reversal of ATIII-dependent fXa inhibition 10 Discussion r-antidote does not interfere with normal fXa function r-antidote with no anticoagulant or procoagulant activity Rapid onset of action & complete reversal of fXa-inhibitors activity Normalises hemostasis from anticoagulant drugs targeting fXa Universal antidote for direct & indirect fXa inhibition Limitations Possible that used in vitro system are not sensitive enough Preclinical data based on animal model, difficulties to extrapolate to humans unidentified interactions with other proteins immunogenicity
Slide 11 - A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa Lu G, Deguzman FR, Hollenbach SJ, et al. Department of Biology, Portola Pharmaceuticals Inc., South San Francisco, California, USA Nature Medicine, April 2013, Volume 19 No 4 pp446 - 451 2 Background Noval oral anticoagulants (factor Xa & IIa inhibitors) widely used Higher incidence for (GI-)bleeding Until now no antidote for nOAC Management of bleeding difficult; Vitamin K, tranexamin acid, FFP, rfVIIa, PCC not effective -> presentation of a recombinant agent antagonizing factor Xa -> proof of concept with in vitro & in vivo investigations 3 Recombinant Antidote (r-antidote) Truncated form of enzymatically inactive fXa Lacks GLA domain, catalytically inactive due to mutation in serine residue Mature functional form, expressed in chinese hamster ovary Aspects to prove: Specific binding to fXa? Reversal of anticoagulation? pro- or antithrombotic properties? Reduce loss of bleeding? 4 r-Antidote: in vitro-essays Reversal of anticoagulation in human plasma: clotting assay & thrombin generation assay: high affinity for fXa-inhibitors Inhibitory activity dose-dependently and completely reversed by r-Antidote no pro- or anticoagulant activity In vivo rat model - reversal of anticoagulation 5 Rapid normalisation of INR after r-antidote infusion Reversal of anticoagulation correlates to reduction of unbound fXa-inhibitor-fraction Mouse model of blood loss (tail transection) ASS+Rivar.+r-antidote reduced blood loss by 85% After r-antidote rivaroxaban activity reduced by >80% 6 Treat-I: vehicle + vehicle + vehicle Treat-II: vehicle + rivaroxaban + vehicle Treat-III: ASA + vehicle + vehicle Treat-IV: ASA + rivaroxaban + vehicle Treat-V: ASA + rivaroxaban + r- Antidote Rabbit model of blood loss (liver laceration model) Laceration of 2 liver lobes and blood loss collection over 15min after injection of vehicle or rivaroxaban and r-antidote 7 PT & aPTT increased 2.3x resp. 1.9x after rivaroxaban, blood loss 3.2x higher r-Antidote reduced blood loss by >85%, decreased anti-fXa activity by 98% Correlation of blood loss with reduction in anti-fXa-activity an free fXa-inhibiton Reversal of ATIII-dependent fXa inhibitors by r-antidote r-antidote mimicking binding-site of fXa for ATIII Enoxaparin (LMWH) & Fondaparinux (pentasaccharide) indirect fXa inhibitor (increase affinity of ATIII for fXa) 8 Fondaparinux Enoxaparin anti-fXa activitiy of enoxaparin & fondaparinux dose-dependently reversed with r-antidote Reversal of ATIII-dependent fXa inhibitors by r-antidote Animal model (rat tail transection) 9 Treat-I: vehicle Treat-II: Enoxaparin & vehicle Treat-III Enoxaparin & r-antidote 2mg/h Treat-IV: Enoxaparin & r-antidote 4mg/h Enoxaparin Treat-I: vehicle Treat-II: Fondaparinux & vehicle Treat-III Fondaparinux & r-antidote Treat-IV: fondaparinux & Protamine Normalisation of hemostasis after r-antidote administration Complete reversal of ATIII-dependent fXa inhibition 10 Discussion r-antidote does not interfere with normal fXa function r-antidote with no anticoagulant or procoagulant activity Rapid onset of action & complete reversal of fXa-inhibitors activity Normalises hemostasis from anticoagulant drugs targeting fXa Universal antidote for direct & indirect fXa inhibition Limitations Possible that used in vitro system are not sensitive enough Preclinical data based on animal model, difficulties to extrapolate to humans unidentified interactions with other proteins immunogenicity 11 Conclusion r-antidote capable to reverse nOAC-induced bleedings Rapid onset of action will have major impact in future daily practice (bleeding management, surgical procedures) Increase safety of using fXa-inhibiting drugs. Phase II-study running Further studies (antidote IIa-inhibitor, unviversal fIIa & fXa-antidote) on the way
 

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