Dabigatran - Rivaroxaban: Variability and monitoring

Dabigatran - Rivaroxaban: Variability and monitoring Jean-Michel Dogné, Jonathan Douxfils, François Mullier, Christian Chatelain, Bernard Chatelain, December 1 st, 2011 1

Anticoagulant landscape 2

Commonly used anticoagulants Parenteral anticoagulants Unfractionated heparin Low molecular weight heparins Indirect Factor Xa inhibitor (fondaparinux( fondaparinux) Oral anticoagulants Vitamin K antagonists Geerts WH et al. Chest 2008;133:381S 453S 3

Traditional anticoagulants: drawbacks UFH 1 Parenteral administration Monitoring and dose adjustment required Risk of HIT LMWH 1 Parenteral administration Weight-adjusted dosing Oral VKAs 2 Narrow therapeutic window Interaction with food and drugs Frequent monitoring and dose adjustment required 1. Hirsh J et al. Chest 2008;133;141S 159S; 2. Ansell J et al. Chest 2008;133;160S 198S 4

The evolution of anticoagulant drugs 1930s 1940s VKAs 1980s LMWHs 1990s Direct thrombin inhibitors 2002 Indirect Factor Xa inhibitor 2004 Oral direct thrombin inhibitors 2008 Oral direct Factor Xa inhibitors Heparin II, VII, IX, X (Protein C, S) AT + Xa + IIa (1:1 ratio) AT + Xa + IIa (Xa > IIa) IIa AT + Xa IIa Xa Perzborn E et al. Nat Rev Drug Discov 2011;10:61-75 5

VKAs target multiple factors in the coagulation pathway Initiation VKA TF VIIa VII VKA X IX Propagation Xa IXa Inactive factor Active factor Transformation Catalysis VKAs inhibit the synthesis of functional coagulation Factors II, VII, IX and X IIa II Prothrombin VKA Thrombin Clot formation Fibrinogen Fibrin Adapted from Spyropoulos AC. Expert Opin Investig Drugs 2007;16:431 440 6

UFH is an indirect inhibitor of Factor Xa and thrombin TF VIIa VII Initiation X IX Propagation Xa IXa Inactive factor Active factor Transformation Catalysis Indirect inhibition by UFH via AT AT IIa II Thrombin Prothrombin Clot formation Fibrinogen Fibrin Adapted from Spyropoulos AC. Expert Opin Investig Drugs 2007;16:431 440 7

LMWH is an indirect inhibitor of Factor Xa and thrombin TF VIIa VII Initiation X IX Propagation Xa IXa Inactive factor Active factor Transformation Catalysis Indirect inhibition by LMWH via AT AT IIa II Thrombin Prothrombin Clot formation Fibrinogen Fibrin Adapted from Spyropoulos AC. Expert Opin Investig Drugs 2007;16:431 440 8

Fondaparinux is an indirect Factor Xa inhibitor TF VIIa VII Initiation X IX Propagation Xa IXa Inactive factor Active factor Transformation Indirect inhibition by fondaparinux via AT AT II Prothrombin Catalysis IIa Thrombin Clot formation Fibrinogen Fibrin Adapted from Spyropoulos AC. Expert Opin Investig Drugs 2007;16:431 440 9

New oral anticoagulants for VTE treatment 10

Direct Factor Xa inhibitors Initiation TF VIIa VII X IX Propagation Xa IXa Inactive factor Active factor Transformation Catalysis Direct Factor Xa inhibition Rivaroxaban Apixaban Edoxaban Betrixaban Darexaban IIa II Thrombin Prothrombin Clot formation Fibrinogen Fibrin Adapted from Spyropoulos AC. Expert Opin Investig Drugs 2007;16:431 440 11

Direct thrombin inhibitors Initiation TF VIIa VII X IX Propagation Xa IXa Inactive factor Active factor Direct Factor IIa inhibition Dabigatran etexilate II Prothrombin Transformation Catalysis IIa Thrombin Clot formation Fibrinogen Fibrin Adapted from Spyropoulos AC. Expert Opin Investig Drugs 2007;16:431 440 12

(R)evolution in anticoagulant therapy AG Turpie; C Esmon. Thromb Haemost 2011; 105: 586 596 13

Main disadvantages of current anticoagulants T. Mavrakanas, H. Bounameaux / Pharmacology & Therapeutics 2011 14

Main disadvantages of current anticoagulants Many patients do not receive satisfactory anticoagulant therapy or stop it too early. Medical need: ideal anticoagulant 15

Properties of «ideal» anticoagulant Proven efficacy (non inferior to current anticoagulant therapy) Oral administration No requirement for routine blood monitoring and dose adjustment Wide therapeutic window Rapid onset of action Predictable pharmacokinetics and pharmacodynamics (good oral availability) Minimal interaction with food and other drugs Ability to inhibit and clot-bound coagulation factors Low non-specific binding Reversibility Availability of an antidote No unexpected toxicities (low bleeding risk,, no hepatic toxicity) Acceptable costs Adapted from T. Mavrakanas, H. Bounameaux / Pharmacology & Therapeutics 2011 and J.Steffel and E.Braunwald European Heart Journal March 2011 16

Potential indications of new oral anticoagulants Prevention of venous thromboembolism -Total hip/knee replacements - Medical ill patients Prevention of stroke and systemic embolism in adult patients with nonvalvular atrial fibrillation Treatment of venous thromboembolism Acute coronary syndrome 17

The promise of new oral anticoagulants Simplified dosing regimen No dietary restrictions Predictable anticoagulation and no need for routine coagulation monitoring Can be given at fixed doses Reduced potential for food and drug interactions Less labour-intensive Less impact on patients daily life Improved compliance Reduced administrative costs Improved quality of life Improved efficacy and safety 1. Raghaven N et al. Drugs Metab Dispos 2009;37:74 81; 2. Shantsila E & Lip GY. Curr Opin Investig Drugs 2008;9:1020 1033; 3. Mueck W et al. Clin Pharmacokinet 2008;47:203 216; 4. Mueck W et al. Thromb Haemost 2008;100:453 461; 5. Mueck W et al. Int J Clin Pharmacol Ther 2007;45:335 344 18

I.Ahrens et al.thromb Haemost 2010; 104: 49 60 19

First new anticoagulant agents Dabigatran etexilate Rivaroxaban 20 20

First new anticoagulant agents Dabigatran etexilate Rivaroxaban Pradaxa Xarelto Competitive, reversible direct thrombin inhibitor EMA authorized: 18/03/2008 Highly selective direct factor Xa inhibitor EMA authorized: 30/09/2008 21 21

Rivaroxaban: : total arthroplasty T. Galanis et al. J Thromb Thrombolysis 2011; 31: 310:320 22

Dabigatran: : total arthroplasty T. Galanis et al. J Thromb Thrombolysis 2011; 31: 310:320 23

Dabigatran: : total arthroplasty -The proposed therapeutic doses (150 or 220 mg q.d.) were found to be non-inferior to enoxaparin in terms of efficacy in the pivotal studies. -There is a trend to higher efficacy of DAB 220mg compared with enoxaparin, associated with an increased bleeding risk. - On the opposite, with DAB 150mg, there is a trend to lower efficacy compared with enoxaparin, associated with a lower bleeding risk risk, which might be useful for some at risk populations (patients with moderate renal impairment, elderly) which have increased DAB exposure. T. Galanis et al. J Thromb Thrombolysis 2011; 31: 310:320 24

Knee replacement surgery Rivaroxaban Hip replacement surgery 10 mg rivaroxaban taken orally once daily. The initial dose should be taken 6 to 10 hours after surgery a treatment duration of 2 weeks is recommended a treatment duration of 5 weeks is recommended 25

Dabigatran Knee replacem ent surgery Hip replacem ent surgery 220 m g once daily taken as 2 capsules of 110 m g Treatm ent should be initiated orally w ithin 1 4 hours of com pleted surgery with a single capsule and continuing w ith 2 capsules once continuing w ith 2 capsules daily thereafter for once daily thereafter for a total of 10 d ays a total of 28-35 days 26

Rivaroxaban Jean-Michel Dogné, Jonathan Douxfils, François Mullier, Christian Chatelain, Bernard Chatelain, 27

Pharmacodynamics Direct, specific, competitive Factor Xa inhibitor Inhibits free and fibrin- bound Factor Xa activity, and prothrombinase activity Inhibits thrombin generation acts earlier in the coagulation cascade No direct effect on thrombin-induced induced platelet aggregation, and thus, on primary haemostasis O O N N O N H O O Rivaroxaban S Cl Perzborn et al., J Thromb Haemost 2005; Pathophysiol Haemost Thromb 2004; Depasse et al., J Thromb Hameost 2005; Kubitza et al., Clin Pharmacol Ther 2005; Br J Clin Pharmacol 2007; Eur J Clin Pharmacol 2005; Graff et al., J Clin Pharmacol 2007; Fareed et al., J Thromb Haemost 2005; Tinel et al., Blood 2006, Roehrig S et al. J Med Chem 2005;48:5900 8 28

Rivaroxaban: High specificity for Factor Xa Inhibition of: Factor Xa Factor VIIa,, Factor XIa,, thrombin, activated protein C, plasmin, urokinase, trypsin Rivaroxaban (nm) K i = 0.4 ± 0.02 IC 50 > 20 000 Rivaroxaban k on = 1.7 10 7 M 1 s 1 E + I E I Rivaroxaban k off = 5 10 3 s 1 k off k on Data from Perzborn E et al. J Thromb Haemost 2005;3:514 21; 21; Tersteegen A et al. J Thromb Haemost 2007;5(Suppl. 2):P-W-651. 29

Rivaroxaban does not require antithrombin as a cofactor Antithrombin activity (median change from baseline) 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 Single dose Healthy volunteers 1.25 mg rivaroxaban (n = 8) 5 mg rivaroxaban (n = 6) 10 mg rivaroxaban (n = 8) 20 mg rivaroxaban (n = 7) 40 mg rivaroxaban (n = 8) 80 mg rivaroxaban (n = 6) Placebo (n = 25) 0.7 0.6 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours) 30 Adapted from Kubitza D et al. Clin Pharmacol Ther 2005;78:412 21, with permission from the Nature Publishing Group.

Rivaroxaban has no significant effect on thrombin activity Thrombin activity (median change from baseline) 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 Single dose Healthy volunteers 1.25 mg rivaroxaban (n = 8) 5 mg rivaroxaban (n = 6) 10 mg rivaroxaban (n = 8) 20 mg rivaroxaban (n = 7) 40 mg rivaroxaban (n = 8) 80 mg rivaroxaban (n = 6) Placebo (n = 25) 0.7 0.6 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours) Adapted from Kubitza D et al. Clin Pharmacol Ther 2005;78:412 21, with permission from the Nature Publishing Group. 31

In vitro human studies In vitro rivaroxaban has no direct effect on platelet aggregation I Platelet aggregation assay Collagen, U46619, ADP, TRAP-6 (human platelet-rich plasma) 1 γ-thrombin (human platelet-rich plasma) 1 Collagen, ADP, epinephrine, arachidonic acid (human platelet-rich plasma, rivaroxaban 23 µm) Collagen 2 (healthy male subjects given rivaroxaban 15 mg) 3 Effect IC 50 >200 µm IC 50 81 µm >75% aggregation No difference from control No change from baseline 1 Perzborn et al., ICT 2004; 2 Fareed et al., ISTH 2005; 3 Kubitza et al., Br J Clin Pharmacol 2007 32

Pharmacokinetics properties E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 33

Pharmacokinetics properties Contraindicated in patient with hepatic disease associated with coagulopathy and an increase of bleeding risks. Is to be used with caution in cirrhotic patients with moderate hepatic impairment (Child Pugh B) if it is not associated with coagulopathy. Risk of hemorrhage. E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 34

Rivaroxaban in patients with hepatic impairment Mild hepatic impairment No relevant differences in PK/PD compared with healthy individuals Moderate hepatic impairment may be associated with coagulopathy Prolonged PT at baseline Increased C max and AUC Effects on PK reflected in PD Moderate prolongation of PT Rivaroxaban plasma concentration (µg/ml) PT (x-fold relative change from baseline) Data from Halabi A et al. J Thromb Haemost 2007;5(Suppl. 2):P-M-635. 350 300 250 200 150 100 50 0 0 4 8 12 16 20 Time (hours) 2.5 2.0 1.5 1.0 0 Rivaroxaban 10 mg Moderate hepatic impairment (n = 8) Mild hepatic impairment (n = 8) Healthy (n = 16) 24 4 8 12 16 20 24 35 Time (hours)

Pharmacokinetics properties Mild renal impairment: (Cl Cr between 50 to 79ml.min -1 ) AUC increase by 44% Moderate renal impairment: (Cl Cr between 30 to 49ml.min -1 ) AUC increase by 52% Severe renal impairment: (Cl Cr <30ml.min -1 ) AUC increase by 54% No recommendation No recommendation, but care is to be taken in patient concomitantly receiving medicinal products which increase rivaroxaban plasma concentration Is to be used with caution and not recommended in patient with Cl Cr <15mL/min Risk of bleeding 36

Phase I: effects of renal impairment on rivaroxaban PK and PD Renal clearance declined with increasing renal impairment PD effects mirrored PK effects Patients with mild and moderate renal impairment have been enrolled in phase II and III trials Data from Halabi A et al. Blood 2006;108(11):Abstract 913; Kubitza et al. Br J Clin Pharmacol. 2010; 70 (5):703-712. Rivaroxaban plasma concentration (µg/l) Prothrombin time (x-fold change from baseline) 250 200 150 100 50 Severe impairment (CrCL < 30 ml/min) Moderate impairment (CrCL 30 49 ml/min) Mild impairment (CrCL 50 79 ml/min) Healthy controls (CrCL 80 ml/min) 0 0 1.8 4 8 12 16 Time (hours) 20 24 1.6 1.4 1.2 1.0 Rivaroxaban 10 mg 0 4 8 12 16 20 24 37 Time (hours)

Pharmacokinetics properties Not recommended in patient receiving concomitant systemic treatment with azoleanti-mycotics (ketoconazole, itraconazole, posaconazole and voriconazole) or protease inhibitor (e.g: ritonavir) E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 38

Pharmacokinetics properties Strong CYP3A4 inducers (rifampicin, phenytoin, carbamazepine, phenobarbital or St. John s Wort) are to be administrated with caution. Reduction of plasma concentration of rivaroxaban Risk of thrombosis E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 39

Influence of CYP3A4 and P-gpP inhibitors on rivaroxaban plasma concentration Rivaroxaban + CYP3A4 inhibition P-gp inhibition AUC x-fold increase C max x-fold increase Clinically relevant Ketoconazole 400 mg o.d. Strong Strong 2.6 1.7 YES* Ritonavir 600 mg b.i.d. Strong Strong 2.5 1.6 YES* Clarithromycin 500 mg b.i.d. Strong Moderate 1.5 1.4 No Erythromycin 500 mg t.i.d. Moderate Moderate 1.3 1.3 No *For full details please see the rivaroxaban summary of product characteristics. Summary of Product Characteristics. http://www.emea.europa.eu/humandocs/pdfs/epar/xarelto/h-944-pi-en.pdf Last accessed: 16/09/09. 40

Pharmacokinetics properties: Interactions E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 41

Phase I: effects of age and gender on rivaroxaban PK and PD No clinically significant effects on exposure or maximum plasma concentrations 1 No clinically relevant differences in PD 1,2 PT closely correlated with plasma concentration 2 Adapted from 1 Kubitza D et al. Poster presented at EFORT 2007; Data from 2 Kubitza D et al. Blood 2006;108(11):Abstract 905. Plasma concentration (µg/l) (geometric mean) Prolongation of PT (relative change from baseline) 150 100 50 1.8 1.6 1.4 1.2 Rivaroxaban 10 mg 300 Young women n = 6 250 Elderly women n = 6 Young men n = 6 200 Elderly men n = 6 0 0 5 10 15 20 25 Time after baseline (hours) Young women n = 6 Elderly women n = 6 Young men n = 5 Elderly men n = 6 Placebo (women) n = 4 Placebo (men) n = 5 1.0 0 5 10 15 20 25 Time (hours) 42

Phase I: effects of body weight on rivaroxaban PK and PD No relevant differences in PK AUC unaffected C max increased by ~24% in subjects 50 kg, not considered clinically relevant Close correlation between PK and PD Rivaroxaban plasma concentration (µg/l) 180 160 140 120 100 80 60 40 20 0 Rivaroxaban 10 mg Body weight 50 kg Body weight 70 80 kg Body weight > 120 kg 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours) Data from Kubitza D et al. J Clin Pharmacol 2007;47:218 26. 43

Rivaroxaban 10 mg o.d.:.: SMPC EU Population Gender Elderly people Body weight Mild/moderate renal impairment Hepatic impairment Fixed dose of rivaroxaban 10 mg o.d.. No dose adjustment. 10 mg o.d.. No dose adjustment. 10 mg o.d.. No dose adjustment. 10 mg o.d. No dose adjustment in mild (CrCL( 50 80 ml/min) or moderate (CrCL( 30 49 ml/min) renal impairment To be used with caution in patients with moderate (CrCL( 30 49 ml/min) renal impairment concomitantly receiving other medicinal products s which increase rivaroxaban plasma concentrations To be used with caution in patients with severe (CrCL( 15 29 ml/min) renal impairment Not recommended in patients with CrCL < 15 ml/min 10 mg o.d. Contraindicated in hepatic disease associated with coagulopathy and clinically relevant bleeding risk. To be used with caution in cirrhotic patients with moderate hepatic impairment (Child Pugh B) if it is not associated with coagulopathy. 44

Why? Situation at risk? Monitoring EMA (SmPC( SmPC) ) recommendations: caution what is the meaning? caution closely monitor patients for decrease effect of rivaroxaban Lack of effectiveness is of major concern Risk of thrombosis How to closely monitor? What are the risk minimizations? Educational material for HCPs and patients? 45

Monitoring Opinion of the regulator agencies EMA (CHMP Report EMEA/543519/2008) There is a need to develop a laboratory test for detecting increased exposure or pharmacodynamic activity. The Applicant has, as a follow-up measure, undertaken to validate modified commercially available tests for estimations of the pharmacodynamic activity of rivaroxaban that could be used in routine clinical setting. 46

Monitoring Why? Situation at risk? Interest of biological monitoring of rivaroxaban Risk of overdose Bleedings Risk of underdose Lack of effectiveness!!! Thrombosis 47

Why?? Situation at risk? Moreover, the Food and Drug Administration mentioned the necessity of a lower strength tablet for the following patients: Child Pugh class B hepatic impairment without coagulopathy Concurrently taking rivaroxaban with a P-gp and strong CYP3A4 inhibitor Concurrently taking rivaroxaban with a P-gp and mild or moderate CYP3A4 inhibitor with mild-moderate renal impairment Need for a comprehensive assessment of the patient to evaluate the risk of bleeding Need for a sensitive, specific and reproducible drug monitoring to assess the impact of all possible interaction. FDA. CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS REVIEW(S). 30-12-2010 48

Situations requiring a biological monitoring Interest of biological monitoring of rivaroxaban Clinical trials Safe and protected environment 49

Situations requiring a biological monitoring Interest of biological monitoring of rivaroxaban Clinical trials Safe and protected environment Monitoring not necessary 50

Situations requiring a biological monitoring Interest of biological monitoring of rivaroxaban Clinical trials Safe and protected environment Real world Monitoring not necessary 51

Situations requiring a biological monitoring Interest of biological monitoring of rivaroxaban Clinical trials Safe and protected environment Real world Monitoring not necessary Monitoring to minimize the risks of bleedings and identify non responders (lack of effectiveness) 52

Monitoring When? At the instauration of the treatment In the case of a switch from AVK to rivaroxaban Relapse of Thombosis or Stroke Bleedings complications To assess compliance 53

Laboratory assays Criteria for a gold-standard Linearity on a wide range of concentration Sensitive Specific Reproducible (CV in %) Rapid Inexpensive Availability 54

Laboratory assays Tests performed with rivaroxaban: Chronometric assays aptt PTT-A CKPrest Cephascreen Actin FS Synthasil 55

Laboratory assays Tests performed with rivaroxaban: Chronometric assays PT Recombiplastin Innovin Neoplastin CI + Neoplastin R Triniclot PT HTF Triniclot PT Excel Triniclot PT Excel S 56

Laboratory assays Tests performed with rivaroxaban: Chromogenic assays STA - Liquid anti-xa (LAX) Hyphen Biomed - Biophen Direct factorxa Inhibitor (Biophen DiXaI) 57

Results: aptt Simulated therapeutic range in AF (32 290ng/mL) aptt is not suitable to evaluate the pharmacodynamic effects of rivaroxaban due to a lack of sensitivity 58

Time (sec) 150 100 50 Results: : PT Recombiplastin (ACLTOP) Innovin (BCS) Neoplastine R (STA) Neoplastine CI+ (STA) Triniclot PT Excel (STA) Triniclot PT HTF (STA) Triniclot PT Excel S (STA) Simulated therapeutic range in AF (32 290ng/mL) 0 0 500 1000 1500 2000 2500 [rivaroxaban] (ng/ml) PT shows a concentration dependent prolongation of clotting time depending on the reagent. 59

Results: : PT Reagent PT value (sec) at baseline PT value (sec) at C trough (32ng/mL) in simulated population in AF Triniclot PT Excel S 16,2 17,5 28,0 PT value (sec) at C max (290ng/mL) in simulated population in AF Recombiplastin 12,1 13,0 20,1 Neoplastin R 15,1 16,0 23,8 Neoplastin CI+ 15,7 16,3 21,4 Triniclot PT Excel 12,5 12,9 16,2 Triniclot PT HTF 14,2 14,7 18,0 Innovin 10,4 10,6 12,4 60

Predictable pharmacodynamics in phase I and II studies Rivaroxaban plasma concentrations correlate closely with its pharmacodynamic c effects, i.e. prothrombin time (only with Neoplastin as thromboplastin) and thrombin generation Prothrombin time (s) 50 40 30 20 10 Healthy subjects (phase I) Observed values Model predictions 0 0 100 200 300 400 500 600 Rivaroxaban plasma concentration (µg/l) Prothrombin time (s) 60 50 40 30 20 10 Patients undergoing major orthopaedic surgery (phase II) 0 0 100 200 300 400 500 600 700 800 Rivaroxaban plasma concentration (µg/l) The relationship between rivaroxaban plasma concentration and prothrombin time was identical in both healthy subjects and in patients undergoing orthopaedic surgery Kubitza et al., Eur J Clin Pharmacol 2005; Graff et al. J Clin Pharmacol 2007; Mueck et al., Clin Pharmacokinet 2008 61

Results: Biophen DiXaI 3 Simulated therapeutic range in AF (32 290ng/mL) 2 OD/mn 1 0 0 500 1000 1500 [rivaroxaban] ng/ml Biophen DiXaI showed a concentration dependent decrease of the OD/min with a linear regression and a good sensitivity. This test is more specific than other anti-xa chromogenic assay because it is performed with a Tris-EDTA-NaCl buffer making it insensitive to the presence of 62 heparin or fondaparinux.

Discussion PT, and Biophen DiXaI are the most appropriate assays to evaluate the pharmacodynamics effects of rivaroxaban on coagulation. Nevertheless, some points are to be considered. 63

Discussion There is a need for a widely available test, giving rapid information (without the necessity of calibration curve and control) about the anticoagulation in case of emergency. Thus, we propose: 1. A screening functional test with PT. 2. If the value of PT exceed a cut-off (to be determined in each lab depending on the reagent used), a confirmation test based on specific chromogenic anti-xa assay should be performed 64

Discussion Importance of standardizing the time between the intake of rivaroxaban and the time of blood collection. Mueck et al. Clin Pharmacokinet 2011; 50(10): 675-686 65

Rapidly absorbed: C max within 2 42 4 hours of oral administration Rivaroxaban plasma concentration (µg/l) 300 250 200 150 100 Single dose Healthy volunteers 1.25 rivaroxaban mg (n = 8) 5 mg rivaroxaban (n = 6) 10 mg rivaroxaban (n = 8) 20 mg rivaroxaban (n = 7) 40 mg rivaroxaban (n = 8) 80 mg rivaroxaban (n = 6) 50 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours) Adapted from Kubitza D et al. Clin Pharmacol Ther 2005;78:412 21, with permission from the Nature Publishing Group. 66

Rapidly absorbed without accumulation Multiple rivaroxaban doses Healthy volunteers Rivaroxaban plasma concentration (µg/l) 400 300 200 100 0 0 1 2 7 8 9 Time (days) Adapted from Kubitza D et al. Eur J Clin Pharmacol 2005;61:873 80, with permission from Springer-Verlag. 5 mg rivaroxaban (n = 7) 10 mg rivaroxaban (n = 7) 20 mg rivaroxaban (n = 7) 30 mg rivaroxaban (n = 8) 67

Discussion Importance of standardizing the time between the intake of rivaroxaban and the time of blood collection. C trough is preferable to C max Concentration are lower, and more stabilized in comparison with the absorption phase where the Cmax is observed. Mueck et al. Clin Pharmacokinet 2011; 50(10): 675-686 68

For PT: Discussion Sensitivity depends on the reagent Results expressed in INR are not applicable (ISI is defined for VKA and are not suitable for rivaroxaban but an ISI may be defined specifically for rivaroxaban to reduce inter-reagent reagent variability) Results have to be expressed in ng/ml,, using a specific calibration curve for each lot (+ controls) (availability of lyophilized plasma containing rivaroxaban) OR Results may be expressed in seconds but specific cut-off depending on the reagent should be defined. [1] Tripodi et al., J Thromb Haemost. 2010. 10.1111/j.1538-7836 69

Discussion For chromogenic assays: Biophen DiXaI: Sensitivity is high for all the tested concentrations with a linear correlation Requires calibration and control Chromogenic assays may be used to evaluate more precisely the anti- Xa effect of the therapy in the patient. 70

Conclusion on monitoring A Two-step approach: First: Screening with PT Define cut-off value (in second; depending on the reagent) for risk of bleeding (overdose) and risk of thrombosis (lack of effectiveness) Measures variable depending on the time between the intake of rivaroxaban and the time of blood collection => High variability INR not acceptable Second: Confirmation with Biophen DiXaI Need for specific calibrator and control to express results in ng/ml Define cut-off value (ng/ml( ng/ml) ) for risk of bleeding (overdose) and risk of thrombosis (lack of o effectiveness) Prothrombin Complex Concentrate may be administrated at the appropriate concentration to counter the excessive anticoagulant activity of rivaroxaban in case of overdose 71

Conclusion on monitoring At the present time, neither directly measured plasma concentrations nor PT prolongation predict bleeding in an individual patient treated with rivaroxaban. There is no rationale (guideline) for changing the timing of administration or the dosing based on laboratory coagulation tests in the routine clinical setting. 72

Invasive procedures and management of bleedings JW. Eikelboom, JI. Weitz. BMJ 2011;342:224-227 73

Neutralization of rivaroxaban A specific antidote that antagonizes the PD effect of rivaroxaban n is not available Activated charcoal to reduce absorption in case of overdose may be considered Should bleeding occur: delay next administration or discontinue treatment as appropriate rivaroxaban has a mean terminal t 1/2 of 7 117 hours appropriate symptomatic treatment (e.g. mechanical compression, surgical interventions, fluid replacement and haemodynamic support, blood product or component transfusion) If life-threatening bleeding cannot be controlled by the above measures, administration of recombinant Factor VIIa may be considered (this recommendation is based on limited non-clinical data) Summary of Product Characteristics. http://www.emea.europa.eu/humandocs/pdfs/epar/xarelto/h-944-pi-en.pdf Last accessed: 16/09/09. 74

Availability of an antidote PCC contains: Factor II, VII,IX and X + protein C and S Was used at 50 U PCC/kg PCC may restore PT value at baseline compared with placebo. Elise S. Eerenberg, et al. Circulation 2011: 124; 1573-1579 75

Dabigatran François Mullier, Jonathan Douxfils, Bernard Chatelain, Christian Chatelain, Jean- Michel Dogné TGG November 23 th, 2011 76

Pharmacology - Pharmacodynamic Primary pharmacodynamic Synthetic, non-peptide, competitive, rapidly acting and reversible inhibitor of active site of thrombin. Inhibits Free thrombin (Ki( of 4.5 nm) Fibrin bound thrombin (observed in a blood clot) Thrombin induced platelet aggregation Secondary pharmacodynamic low affinity (Ki>3.5 µm) towards the serine proteases factor Xa,, factor XIa,, factor VIIa/tissue factor complex,, plasma kallikrein, plasmin, urokinase, tissue-type type plasminogen activator, activated protein C, granulocyte elastase and C1 esterase. inhibits trypsin with a Ki of 50.3 nm, 77

Pharmacology - PK H 3 C O O Oral prodrug N N O CH 3 N N HN O NH 2 N PRADAXA = Dabigatran etexilate O CH 3 Gastrointestinal absorption HO O Active metabolite in plasma N N O CH 3 N N HN NH 2 NH Dabigatran 78

Pharmacology - PK Low with a very large interindividual variability of PK parameters (Cmax, AUC). the interindividual variability of Cmax and AUC expressed as CV was high i.e. approximately 80%. In healthy volunteers the intraindividual variability was close to 30%. E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 79

Pharmacology - PK Low with a very large interindividual variability of PK parameters (Cmax, AUC). Risks of underdosage (thrombosis) or overdosage (bleeding) E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 Importance of monitoring and individual follow-up 80

Pharmacology - PK moderate renal insufficiency (CrCL between 30 50 ml/min): 2.7-fold AUC increase Pradaxa should be used with caution A close clinical surveillance (looking for signs of bleeding or anemia) E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 recommended dose is 150 mg taken once daily as 2 capsules of 75 mg (in stead of 220 mg: 2x110mg) 81

Pharmacology - PK Severe renal deficiency (CrCL between 10 30 ml/min): 6-fold AUC increase => CONTRA-INDICATED E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 82

Pharmacology - PK Plasma concentrations of dabigatran might be elevated when co-administered with strong P-gp inhibitors: verapamil, amiodarone. This may increase the risk of bleeding and these patients should be closely Clinically monitored (looking for signs of bleeding and anaemia) E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 83

Pharmacology - PK P- glycoprotein inhibitors: Caution should be exercised with strong P- glycoprotein inhibitors.. The P-P glycoprotein inhibitor quinidine is contraindicated. P- glycoprotein inducers: Potent P- glycoprotein inducers such as rifampicin or St John s wort (Hypericum perforatum), may reduce the systemic exposure of dabigatran. Caution is advised when co-administering these medicinal products. 84

E Nutescu et al. ; J Thromb Thrombolysis 2011; 31: 326-343 85

Who said no monitoring? EMA public assessment report: Pradaxa in the prevention of Venous Thromboembolism (VTE) in patients following elective knee or hip replacement surgery Conclusion on balance benefit-risk in the indication It is important to underline that the PK characteristics of DAB i.e low bioavailability (6.5%) with a very large interindividual variability, the concentration-effect effect relationship and the bleeding risks strongly suggest that drug monitoring is needed. Based on the above balance the benefits associated with the proposed osed use of DE are considered to outweigh the risks. 86

Who said no monitoring? EMA public assessment report: Pradaxa in the prevention of Venous Thromboembolism (VTE) in patients following elective knee or hip replacement surgery Follow-up measure The possibility of drug monitoring would be valuable, especially for these patients at risk and commercially available TT test kits will be developed for measurement of TT following administration of DTIs. A A cross-validation of chronometric TT measurements at local laboratories will be performed as part of the FUM. Commercially available TT test kits developed for measurement of TT following administration of DTIs (i.e. hirudin) ) will be validated for measurement of TT of DAB. 87

[ ] In patients at high risk of bleeding a reduction in dabigatran dose may be necessary. A diluted Thrombin Time test (dtt) is commercially available and can be used to identify patients at increased risk because of excessive exposure to dabigatran, e.g. when renal function could be impaired [ ]. 88

How to monitor? APTT: Activated Partial Thromboplastin Time PT: Prothrombin Time dpt:dilute Prothrombin Time TT: Thrombin Time PiCT: Prothrombinase induced Clotting Time ECT: Ecarin Clotting Time ECA-T: Ecarin Chromogen Assay ACT: Activated Clotting Time Hemoclot Thrombin Inhibitor assay (Hyphen BioMed) HepTest antixa chromogenic assays (StaChrom( and Rotachrom/ / Liquid anti Xa) Thrombin Generation test (TGT) Thromboelastogram (TEG) 89 89

aptt As recently recommended by Australian regulatory authorities, aptt could be used as a screening test to exclude a bleeding risk associated with DE administration. Douxfils J. Mullier F et al. submitted 90

Dabigatran: APTT Australian guideline Douxfils J. Mullier F et al. submitted 91

Hemoclot : Thrombin Inhibitor assay Sensitive diluted TT assay which allows for quantitative measurement of DTI activity in plasma, based on inhibition of a constant and defined concentration of thrombin. Diluted test plasma (1:8 to 1:20) is mixed with normal pooled human plasma, and clotting is then initiated by adding a constant amount of highly purified human α-thrombin. Calibrators and controls available since december 2010. 92 92

Hemoclot : Thrombin Inhibitor assay Douxfils J. Mullier F et al. submitted 93 93

Hemoclot : Thrombin Inhibitor assay HTI: - One of the most sensitive with a 2 x CT of 8ng/mL - Reproducibility was quite good (1.0%). - Linear correlation coefficient (R 2 =1.00) - Addition of NPP (reagent 1) insensitive to fibrinogen. - In a patient taking 150mg of DE bid, a concentration higher than 200ng/mL is correlated with an increased risk of bleeding while this value is reduced to 67ng/mL for patient in primary prevention of venous thromboembolism. Douxfils J. Mullier F et al. submitted 94 94

Practical approach For patient taking 150mg DE bid regimen, an aptt above 80 seconds at trough (corresponding to a Ctrough about 200ng/mL) is correlated with an increased risk of bleeding. This cut-off is reduced to 45 seconds (corresponding to a Ctrough of 67ng/mL) for patients taking 220mg DE qd as for primary prevention of venous thromboembolism. Nevertheless, first, as illustrated in our study, these cut-offs have to be adapted according to the aptt reagent: The clotting time related to a concentration of 200ng/mL varies from 48.6 to 62.5 sec, according g to the reagent. Accordingly, the aptt ratio for this concentration ranges from 1.74 to 2.06. These differences may lead to a misinterpretation if inappropriate cut- offs are used. Australian regulatory authorities proposed an APTT T cut- off of 80 sec based on a concentration of 200ng/mL. Douxfils J. Mullier F et al. submitted 95

Delay Standardization of the time between the last intake of dabigatran and the time of blood collection as these influence dabigatran concentration and thus the results of the coagulation assay. Which? - Ctrough may be more interesting than Cmax since the absorption phase and Cmax are more variable than Ctrough. - Nevertheless, if Ctrough could be used to measure a risk of bleeding with accurate define cut-off, it seems unappropriate to evaluate compliance. - Indeed, the range of local normal values for different aptt showed minor differences in comparison with Ctrough in AF. Moreover, in real-life, life, the baseline clotting time (before drug administration) will often not n be determined precluding to detect a relative change in aptt.. Thus, in this case, more specific and sensitive assays should be used. Douxfils J. Mullier F et al. submitted 96

Discussion: Stangier et al. Br J Clin Pharmacol. 2007;64(3):292-303. 97

Practical approach Reagent Local normal values (sec) Baseline time (sec) Coagulation time of NPP corresponding to mean C trough in AF (i.e 80ng/mL) Coagulation time corresponding to a risk a bleeding in MOS (i.e 67ng/mL) Coagulation time corresponding to a risk a bleeding in AF (i.e 200ng/mL) Sec Ratio Sec Ratio Sec Ratio Actin FS 25.8-33.2 30.3 46.3 1.53 44.3 1.48 62.5 2.06 Cephascreen N.D 27.4 37.5 1.37 36.2 1.32 48.6 1.77 CKPrest 26.7-37.6 30.5 41.6 1.36 40.2 1.32 53.0 1.74 PTT-A 28.0-39.0 33.2 45.2 1.36 44.0 1.32 58.6 1.77 Synthasil 25.8-33.2 27.5 38.0 1.39 36.7 1.33 49.0 1.78 Hemoclot N.D 33.3 42.3 1.27 40.9 1.23 54.7 1.64 Douxfils J. Mullier F et al. resubmitted 98

Practical approach However, using 5 different reagents, the aptt was lower than 80 sec for this concentration in dabigatran.. In addition, the response of an aptt reagent varies according to the lot number and coagulometers showed differences in end point detection. Therefore, each laboratory should therefore calibrate each lot of its aptt reagent on one instrument by spiking a NPP with at least 6 dabigatran concentrations ranging from 0 to 1,000ng/ml. In one particular lab, the aptt is also affected by preanalytical (time of blood sampling and sample transport) and biological variables (lupus anticoagulant, a hereditary or acquired factor deficiencies) (37) Douxfils J. Mullier F et al. resubmitted 99

Practical approach aptt > cut-off HTI. Ratio for HTI < aptt but - Highly reproducible - Linear on a broad range of concentrations in comparison with aptt showing a loss of sensitivity for higher concentration. - Calibrators and specific methodologies available from Hyphen Biomed to easily perform these measurements on BCS,, ACL 7000,, ACL Top, STA-R and provisory on Sysmex CA1500. - As aptt and HTI are global assays, it is also necessary for the clinical biologist to know which anticoagulant is administrated to choose the adequate assay with accurate normal ranges. Douxfils J. Mullier F et al. resubmitted 100

Comments Validation in patients receiving Pradaxa.. Indeed, it is currently unknown how coagulation tests are predictive of the bleeding risks (39( 39). However, Freyburger et al. - explored the impact of DE on patients undergoing THR or TKR (16). - correlation with those obtained in vitro. - used a dilute thrombin time based on the same principle than HTI to assess the impact of dabigatran on the coagulation. - Interindividual variability value of point measurement to minimize the risk of the product. 101

Renal function EMA recommendations following an evaluation of reports of 6 cases s of fatal bleeding in Japan (October 2011) Most patients that experienced fatal bleeding in Japan were elderly with severe renal impairment. In the current EU SmPC for Pradaxa it is stated that factors such as high age, moderate renal impairment (30-50 ml/min CrCL), low body weight, use of acetylsalicylic acid, clopidogrel or NSAID, and presence of esophagitis/gastritis/gastroesophageal reflux requiring treatment increase the risk of bleeding associated with Pradaxa treatment. Furthermore, patients at increased risk of bleeding should be closely clinically monitored looking for signs of bleeding and anaemia. 102

Renal function EMA recommendations following an evaluation of reports of 6 cases s of fatal bleeding in Japan (October 2011) Prior to initiation of treatment with Pradaxa the renal function should be assessed by calculating the creatinine clearance (CrCl( CrCl) ) to exclude patients for treatment with severe renal impairment (i.e. CrCl < 30 ml/min). While on treatment renal function should be assessed in certain clinical situations when it is suspected that the renal function could decline or deteriorate (e.g. hypovolemia,, dehydration, and with certain comedications). In patients above 75 years of age or in patients with renal impairment irment renal function should be evaluated at least yearly. 103

Conclusions New oral anticoagulants: Developed with the intent not to require monitoring due to their predictable pharmacologic effects However this will be required in some specific settings (liver, kidney, drug interactions, compliance, bleeding, recurrence) Therapeutic ranges for coagulation not delineated evaluation of a bleeding complication will be a challenge 104 104

Conclusions Widespread use, 24h/24 accessibility, low cost and relatively good sensitivity aptt could be used for point measurements of dabigatran and as screening test for the risk of bleeding. HTI, ECT and TGA are the most sensitive tests. Besides, HTI showed good reproducibility, excellent linear correlation at all doses, simplicity of use, automation capabilities and should therefore be seen today as the gold-standard assay for point measurements of dabigatran after a positive screening test. 105 105

Conclusions Strategies - APTT followed by Hemoclot - Hemoclot Other drugs - Which drug - Delay: which one? Standardisation! All of these issues will be encountered in a near future the implementation of these antithrombotic agents will require a multi-modal modal team approach and diligent post-approval monitoring for effectiveness and safety 106 106

Management of bleedings on dabigatran Van Ryn et al. Dabigatran etexilate a novel, reversible, oral direct thrombin inhibitor: Interpretation of coagulation assays and reversal of anticoagulant activity.thromb.haemost 2010; 104: 49 60 107

Management of bleedings on dabigatran Van Ryn et al. Dabigatran etexilate a novel, reversible, oral direct thrombin inhibitor: Interpretation of coagulation assays and reversal of anticoagulant activity.thromb.haemost 2010; 104: 49 60 108

Thank you for your attention! Jean-Michel Dogné, Jonathan Douxfils, François Mullier, Christian Chatelain, Bernard Chatelain, 109

Back up slide 110

Practical management: frequently asked questions

Practical management: laboratory monitoring Rivaroxaban does not require routine coagulation monitoring The following coagulation tests are influenced after administration of rivaroxaban: PT, aptt and calculated INR INR testing was developed for measuring VKA effects and is, therefore, not appropriate to measure activity of rivaroxaban If clinically indicated, haemostatic status can be assessed by PT P using Neoplastine as described in the Summary of Product Characteristics Rivaroxaban calibrators and controls are currently under validation and development Anti-Factor Xa assays are also currently under development Xarelto Summary of Product Characteristics 112

Practical management: switching drugs Switching from VKAs to rivaroxaban VKA treatment should be stopped and rivaroxaban should be initiated ted when the INR is 2.5 INR measurement is not appropriate to measure rivaroxaban and should not be used; treatment with rivaroxaban only does not require routine coagulation monitoring Switching from a parenteral anticoagulant to rivaroxaban Rivaroxaban should be started 0 20 2 hours before the time of the next scheduled administration of the parenteral medicinal product (e.g. LMWH) or at the time of discontinuation of a continuously administered parenteral medicinal product (e.g. i.v. UFH) Switching from rivaroxaban to parenteral anticoagulants Give the first dose of parenteral anticoagulant at the time the next rivaroxaban dose would be taken Xarelto Summary of Product Characteristics 113

Practical management: dosing before and after invasive procedures Rivaroxaban should be stopped at least 24 hours before the intervention, if possible, and based on the clinical judgement of o the physician If the procedure cannot be delayed the increased risk of bleeding should be assessed against the urgency of the intervention Rivaroxaban should be restarted after the invasive procedure or surgical intervention as soon as possible, provided the clinical situation allows and adequate haemostasis has been established Xarelto Summary of Product Characteristics 114

Clinical utility of rivaroxaban for treatment of DVT and secondary prevention of VTE Does not require injection or routine coagulation monitoring Rapid anticoagulant effects (within 2 42 4 hours) High oral bioavailability Low potential for drug drug or food drug drug interactions Enables single-drug approach versus LMWH plus warfarin/vka for the initial treatment of VTE and a convenient once-daily dosing for long-term treatment and prevention of VTE recurrence The EINSTEIN Investigators. N Engl J Med 2010;363:2499 2500; Xarelto Summary of Product Characteristics 115

Need for monitoring according to guidelines VKAs Unpredictable anticoagulation response that necessitates regular monitoring 1,2 PT/INR testing to ensure patients are within INR range 2,3 LMWHs Routine coagulation monitoring not normally needed, except for patients with severe renal failure and pregnant women 2 Anti-Factor Xa assay can be used 2 UFH Anticoagulant response varies among patients; UFH (intravenous and subcutaneous) requires monitoring (weight-based subcutaneous UFH does not) 2,4 aptt test to be used to maintain doses that correspond to therapeutic eutic heparin levels 2 1. Merli G et al. Ann Surg 2009;250:219 228; 2. Kearon C et al. Chest 2008;133:454S 545S; 3. Ansell J et al. Chest 2008;133:160S 198S; 4. Hirsh J et al. Chest 2008;133:141S 159S 116