Reversal of New Oral Anticoagulants

Reversal of New Oral Anticoagulants http://www.keepcalm-o-matic.co.uk April Hinds, PharmD PGY2 Ambulatory Care Resident Blackstock Family Practice/Community Care Clinic The University of Texas College of Pharmacy at Austin October 17, 2014 Learning Objectives: 1. Describe new oral anticoagulants place in therapy 2. Explain the mechanism of action and compare differences between new oral anticoagulants 3. Assess bleeding risk of new oral anticoagulants 4. Summarize current available evidence for the reversal of new oral anticoagulants 5. Recommend agents for reversal of new oral anticoagulants Hinds 1

I. New oral anticoagulants: place in therapy A. Epidemiology 1,2 i. Deep venous thrombosis/pulmonary embolism (DVT/PE) 1. DVT: formation of a thrombus in the vein 2. PE: when a thrombus is dislodged into the pulmonary circulation and occludes the pulmonary arteries 3. Approximately 300,000-600,000 affected annually in the U.S. 4. Venous thrombosis frequency increases with age a. As high as 1 in 100 in those >80 years old ii. Atrial fibrillation 3,4,5 1. Structural/electrical abnormalities that change atrial tissue and cause abnormal impulse formation 2. Most common type of arrhythmia 3. Estimated occurrence 2.66 million people in 2010 4. Incidence increases with age a. 1 in 4 adults 40 years b. Median age of atrial fibrillation diagnosis: 66.8 years for men, 74.6 years for women 2. CHADS 2 score used for stroke risk stratification ii. DVT prophylaxis in knee/hip replacement surgery 6 1. 35 day untreated baseline risk for DVT is approximately 4.3% iii. Morbidity and mortality 1,2,5 1. Approximately 60,000-100,000 Americans die of DVT/PE annually a. 10-30% die within one month of DVT diagnosis b. Sudden death occurs in 25% of PE patients 2. One-half of PE patients have long-term complications 3. One-third of patients have a recurrent DVT within 10 years 4. Nonvalvular atrial fibrillation can lead to stroke and heart failure a. Atrial fibrillation causes 15-20% of ischemic strokes b. Stroke incidence increases based on CHADS 2 score B. New oral anticoagulants: apixaban, dabigatran, rivaroxaban Table 1: FDA Approved Indications for New Oral Anticoagulants 7,8,9 FDA Indications Dabigatran Rivaroxaban Apixaban DVT/PE treatment and reducing risk of recurrent DVT/PE after initial therapy Stroke prevention in nonvalvular atrial fibrillation DVT prophylaxis following replacement surgery of hip or knee X X X X X X X Hinds 2

Figure 1: Clotting Cascade and New Oral Anticoagulants 10 II. i. New oral anticoagulants (NOACs) versus warfarin therapy 11 1. Reduction in intracranial hemorrhage 2. Bleeding rates similar or lower (0.1-5.6%) 3. More dependent on renal excretion 4. Fixed doses 5. No routine monitoring 6. No major food interactions Comparison of new oral anticoagulants: metabolism and bleeding risk A. Comparison of agents Table 2: Comparison of New Oral Anticoagulants 7,8,9 Dabigatran Rivaroxaban Apixaban Target Factor IIa (direct Factor Xa Factor Xa thrombin inhibitor) Prodrug Yes No No Bioavailability 6.5% >80% 50% Half-life 14-17 hours 5-9 hours 10-14 hours Metabolism Hepatic Hepatic Hepatic Elimination 80% renal 66% renal 27% renal Plasma protein binding 34-35% 92-95% 87% Drug interactions P-gp inducers/ inhibitors P-gp inducers/ inhibitors, 3A4 P-gp inducers/ inhibitors, 3A4 P-gp: P-glycoprotein; see Appendix A for additional information, pg 18 B. Bleeding risk of new oral anticoagulants i. Dabigatran 12 1. Statistically significant higher bleeding rates for life-threatening bleeding and minor bleeding in warfarin group versus dabigatran 2. Statistically significant higher bleeding rates for gastrointestinal bleeding in dabigatran group versus warfarin 3. No significant difference between bleeding rates for intracranial hemorrhage or major bleeding in dabigatran group versus warfarin Hinds 3

Table 3: Bleeding Events in Dabigatran vs Warfarin 12 Dabigatran 150 Warfarin mg twice daily (n=6022) (%) (n=6076) (%) Relative Risk (95% CI) P-value Intracranial hemorrhage 36 (0.59) 87 (1.44) 0.40(0.27-0.60) 0.38 -- Gastrointestinal 182 (3.0) 120 (1.99) 1.50 (1.19-1.89) <0.001 99 Life-threatening bleeding 175 (2.88) 212 (3.52) 0.81 (0.66-0.99) 0.04 156 Major bleeding 375 (6.17) 397 (6.59) 0.93 (0.81-1.07) 0.31 -- Minor bleeding 1787 (29.4) 1931 (32.0) 0.91 (0.85-0.967) 0.005 38 Major bleeding: hemoglobin reduction 20 gm/liter, transfusion 2 units blood, symptomatic bleeding in a critical area or organ; life-threatening bleeding: fatal bleeding, symptomatic intracranial bleeding, bleeding with a decrease in hemoglobin 50 gm/liter, bleeding requiring 4 units blood or inotropic agent for necessitating surgery; risk of major bleeding increased with age 75 years (HR 1.2, CI 1-1.4); NNH: number needed to harm ii. Apixaban 13 1. Statistically significant lower rates of major bleeding, intracranial bleeding, and any bleeding in apixaban group compared to warfarin 2. No statistical difference between gastrointestinal bleeding rates in the apixaban group compared to warfarin Table 4: Bleeding Events in Apixaban Patients with Nonvalvular Atrial Fibrillation 13 Apixaban 5 mg twice Warfarin Hazard Ratio P-value NNH daily (n=9088) (%) (n=9052) (%) (95% CI) Major Bleeding 327 (3.6) 462 (5.1) 0.69 (0.60-0.80) <0.001 66 Gastrointestinal 105 (1.16) 119 (1.31) 0.89 (0.70-1.15) 0.37 -- Intracranial 52 (0.57) 122 (1.35) 0.42 (0.30-0.58) <0.001 128 Any bleeding 2356 (25.9) 3060 (33.8) 0.71 (0.68-0.75) <0.001 12 Major bleeding: decrease in hemoglobin of 2 gm/dl or transfusion 2 units packed red cells, occurring in a critical site or resulting in death iii. Rivaroxaban 14 1. No statistically significant difference between rivaroxaban and enoxaparin/warfarin bleeding rates Table 5: Bleeding Events in Rivaroxaban Patients in the Treatment of Symptomatic Venous Thromboembolism 14 Rivaroxaban Enoxaparin/warfarin Hazard Ratio P-value (n=1731) (%) (n=1718) (%) (95% CI) Major bleeding 14 (0.8) 20 (1.2) 0.65 0.21 (0.33-1.3) Clinically relevant nonmajor bleeding 126 (7.3) 119 (7.0) -- -- First major or clinically relevant 139 (8.1) 138 (8.1) 0.97 0.77 nonmajor bleeding occurring during treatment (0.76-1.22) Major bleeding: decrease hemoglobin 2 gm/dl and/or transfusion 2 units blood or both, bleeding in a critical site, contributing to death NNH Hinds 4

III. IV. Clinical question A. Given the risk of bleeding with new oral anticoagulants, how can they be reversed? Reversal of new oral anticoagulants A. Reversal strategies in prescribing information i. Apixaban 8 1. No established reversal 2. Effects lasting at least 24 hours after last dose 3. Use of prothrombin complex concentrate (PCC), activated prothrombin complex concentrate (apcc), or recombinant factor VIIa (rfviia) may be considered (no evaluation in clinical studies) 4. Activated charcoal reduces absorption of apixaban ii. Dabigatran 7 1. No specific reversal agent 2. Hemodialysis removes dabigatran but limited clinical experience in use 3. Use of apccs, rfviia, or concentrates of coagulation factors II, IX, or X may be considered (no clinical trial data) 4. If bleeding complications occur, stop dabigatran, provide clinical support, and look for source of bleeding iii. Rivaroxaban 9 1. No specific antidote 2. Partial reversal of prothrombin time prolongation with PCC administration in healthy volunteers 3. Use of apcc or rfviia not evaluated 4. Consider activated charcoal in overdose B. Supportive measures 15,16,17 i. Observation and withdrawal of further anticoagulant therapy ii. Activated charcoal if <2 hours since administration of NOAC iii. Hemodialysis 1. Dabigatran only (least protein bound agent) 2. Removes ~60% of the drug (48.8%-68% at 4 hours) iv. Mechanical compression C. Reversal strategies i. Fresh frozen plasma (FFP) 18 1. Plasma transfusion 2. Contains coagulation factors in normal serum concentrations a. Significantly less coagulation factors when compared to PCCs 3. Mechanism of action: restores all coagulation factors 4. Limitations: must be thawed, ABO matching required, large volume of administration a. Dose: 10-15 ml/kg 5. Adverse effects: fluid overload, infectious disease, transfusion related acute lung injury, allergic reactions, risk of thromboembolism 6. Reduced intracranial hemorrhage volume in a mouse model after administration of dabigatran ii. 3-factor prothrombin complex concentrates (3F-PCC) 16,17 1. Similar concentrations of nonactivated factors II, IX, and X but low concentrations of nonactivated factor VII a. Some formulations may contain additional agents/proteins Hinds 5

2. Proposed mechanism of NOAC reversal: large amounts of factor X may decrease inhibitory effects of factor Xa inhibitors; may overcome factor Xa inhibition by increasing thrombin generation 20 3. Only 2 available in the US (Bebulin VH and Profilnine SD) 4. 3F-PCC less likely to increase thrombosis than 4F-PCC a. 0.7% reported incidence of thromboembolic events with 3F-PCC versus 1.8% in 4F-PCC in reversal of warfarin 21 5. Warning: risk of transmitting infectious agents is possible 6. Bebulin VH: factor IX complex nanofiltered and vapor heated 21 a. Indication: control/prevention of bleeding episodes in adults with hemophilia B (congenital factor IX deficiency) b. Contains factor II, IX, X, low amounts of factor VII and heparin c. Contraindications: allergy to heparin, history of HIT (heparin induced thrombocytopenia) d. Adverse effects: hypotension, dizziness, urticaria, erythema, pyrexia, chills, DVT, PE, stroke, disseminated intravascular coagulation (DIC) 7. Profilnine SD: factor IX complex solvent detergent treated 22 a. Indication: control and prevention of bleeding in factor IX deficient patients due to hemophilia B b. Contains factor II, IX, X, and low levels factor VII c. Adverse effects: urticaria, nausea, vomiting, headache, fever, chills, somnolence, thrombosis, DIC i. Infrequent but consistent reports have been described which indicate that patients are at greater risk of developing thrombosis 22 iii. 4-factor prothrombin complex concentrates (4F-PCC) 16 1. Similar concentrations of factors II, VII, IX, and X a. Contains higher concentrations of factor VII compared to 3F- PCCs b. Some formulations contain additional coagulants c. Available in activated and nonactivated factor VII 2. Warning: risk of transmitting infectious agents is possible 3. Kcentra 16,23 a. Non-activated 4F-PCC contains 25 times more concentrated coagulation factors than found in the plasma b. Indication: urgent reversal of acquired coagulation factor deficiency induced by vitamin K antagonist in acute major bleeding or need for urgent surgery/invasive procedure c. Contains factors II, VII, IX, X, proteins C and S, antithrombin III d. Dose: based on INR values and weight e. Mechanism of action: rapidly increases plasma levels of vitamin K-dependent coagulation factor II, VII, IX, and X and proteins C/S f. Contraindicated: DIC, HIT g. Adverse effects: i. Most frequent: headache, nausea, hypotension, anemia ii. Most severe: stroke, PE, DVT, increased risk of thrombosis iii. Thromboembolic events in acute major bleeding study (8.7%): stroke (1.9%), venous calf thrombosis (1%), DVT (1%), fistula clot (1%) Hinds 6

4. FEIBA 24 a. Activated 4F-PCC b. Indication: treatment of spontaneous bleeding episodes or surgical interventions in hemophilia A and B patients c. Contains factors II, IX, X, and activated factor VII d. Mechanism of action: rapidly increases plasma levels of vitamin K-dependent coagulation factors II, VII, IX, and X (see Appendix B, pg 19) e. Contraindicated: normal coagulation mechanism, treatment of bleeding episodes resulting from coagulation factor deficiencies in the absence of inhibitors to coagulation Factor VIII or IX, DIC, acute thrombosis or embolism f. Adverse effects: thrombosis, somnolence, dizziness, nausea, pyrexia, chills, bleeding in nonhemophiliac patients i. Rate of thrombosis: 4-9 per 100,000 infusions 25 1. Dose dependent side effect iv. Recombinant factor VIIa (rfviia) 1. NovoSeven 16,26 a. Indication: Peri-operative management and treatment of bleeding episodes in adults/children with hemophilia A or B with inhibitors; congenital factor VII deficiency or acquired hemophilia; Glanzmann s thrombasthenia with refractoriness to platelet transfusions b. Contains activated factor VII c. Mechanism of action: after complexing with tissue factor (TF), activated coagulation factor X to factor Xa and factor IX to factor IXa; factor Xa then converts prothrombin to thrombin and forms a hemostatic plug via the conversion of fibrinogen to fibrin d. Adverse effects: thrombosis, bleeding i. Black box warning: can cause arterial and venous thrombotic events ii. 4% occurrence of thromboembolic events (cerebral artery occlusion, cerebral ischemia, angina pectoris, myocardial infarction, PE, DVT) Brand Names 3F-PCCs Bebulin VH Factors II, IX, X, low concentration factor VII, heparin Profilnine SD Table 6: PCCs available in the US 21,22,23,24,25,26 Components Dose Time to effect (min) Factors II, IX, X, low concentration factor VII Duration (hr) Cost (AWP) 25-70 units/kg 10 6-8 $1.14/ IU 1 unit/kg will increase Factor IX 1% 10 6-8 $1.19/ IU Activated 4F- PCC FEIBA NF Factors II, VII, IX, X, 50-100 units/kg 15 8-12 $2.17/ IU 4F-PCC Kcentra Factors II, VII, IX, X, protein C/S, 25-50 units/kg 15 6-8 $2.17/ IU antithrombin III rfviia NovoSeven Activated rfviia 15-90 mcg/kg 10 < 1 $1.64/ mcg AWP: average wholesale price Hinds 7

Table 7: PCC Components (units/ml) 21,22,23,24,26 Bebulin VH Profilnine SD FEIBA NF Kcentra* Factor II 24-38 35-40 1.3 units/feiba unit 19-40 Factor VII <5 10 0.9 units/feiba unit 10-25 Factor IX 24-38 25 1.4 units/feiba unit 20-31 Factor X 24-38 25 1.1 units/feiba unit 25-51 Heparin <0.15 U/U Factor IX -- -- 0.4-2 *Kcentra also contains proteins C/S and antithrombin III; heparin theorized to decrease risk of thrombosis D. Drugs on the horizon i. Andexanet alfa (r-antidote) 27 1. Catalytically inactive, recombinant protein, binds to direct factor Xa inhibitors and activated antithrombin III a. Dose dependently reverses the inhibition of factor Xa inhibitors 2. Phase 1 trial, 32 healthy volunteers randomized to receive various single IV bolus doses of andexanet alfa (30, 90, 300, or 600 mg) or placebo 28 a. Rivaroxaban added to plasma samples ex vivo b. Anticoagulant effect reversed in a dose-dependent fashion c. No thrombotic events/deaths reported over 28 day follow up 25 3. Currently in phase 2 and 3 trials 29,30 a. Serious reported adverse effects: 1 case of pneumonia b. Administration of andexanet alfa IV bolus followed by a 2 hour continuous infusion after the administration of apixaban produced a rapid, nearly complete, and sustained reversal of the anticoagulant effect c. Ongoing phase 2 proof-of-concept study interim results for 6 day treatment with apixaban 5 mg twice daily (n=6), after a 420 mg bolus andexanet alfa; anticoagulant reversal of apixaban was 92% (p<0.0001) compared to placebo (n=3), and after the two hour infusion, reversal was 91% (p<0.0001) d. 210 mg and 420 mg of andexanet alfa reversed rivaroxaban s anticoagulant effects (20 mg daily) by 32% and 51% after 2 minutes, respectively ii. PER977 31 1. Synthetic small molecule antidote for potential reversal of apixaban, rivaroxaban, dabigatran, fondaparinux, low molecular weight heparin 2. Demonstrated ex vivo reversal of apixaban and rivaroxaban when analyzing aptt and anti-xa levels iii. adabi-fab 32 1. Antibody fragment for reversal of dabigatran 2. Mimics thrombin, high affinity for dabigatran 3. Reversed dabigatran clotting time in an in vivo rat model Hinds 8

V. Evidence for Reversal of New Oral Anticoagulant Table 8: Perzborn E, Heitmeier S, Laux V, Buchmuller A. Reversal of rivaroxaban-induced anticoagulation with prothrombin complex concentrate, activated prothrombin complex concentrate and recombinant activated factor VII in vitro. Thromb Res. 2014;133:671-681. 33 Purpose To determine the potential of PCC, apcc, and rfviia to reverse the anticoagulant activity of rivaroxaban in human blood Design In vitro study Inclusion Healthy subjects Exclusion Medications in previous 10 days Outcomes Reversal of anticoagulant effects by measuring prothrombin time (PT), clotting time (CT), thrombin generation (TG), lag time, maximum concentration of TG (C max), endogenous thrombin potential (ETP) (see Appendix D, pg. 20) Methods Rivaroxaban was diluted to a concentration to simulate a 20 mg dose and hypothetical overdoses and added to blood samples from healthy subjects; test conducted for 1 hour Reversal agents were also diluted to simulate reasonable human plasma concentrations apcc (FIEBA) 0.2 U/mL, 0.4 U/mL, 0.7 U/mL (50 U/kg), 1 U/mL (50-100 U/kg) 4F-PCC (Beriplex) 0.4 U/mL (25 U/kg), 0.2 U/mL, 0.7 U/mL (50 U/kg) (comparable to Kcentra factor concentrations) rfviia (Novoseven) 5 μg/ml (270 μg/kg), 15 μg/ml, 50 μg/ml Statistics Analysis of variance (ANOVA) and Tukey s multiple comparison test Results Rivaroxaban 200-1000 ng/ml prolonged PT in human plasma by 2.1 to 5.5 fold from baseline in a concentration dependent fashion Prothrombin time (PT) PCC: decreased PT by 15-22% from 13.1 seconds to 12.3 seconds (not significant at 200 ng/ml, n=7); reversal significant at 500 ng/ml and 1000 ng/ml (P<0.001 vs rivaroxaban) apcc: significantly shortened PT by 22-24% from 12.8 seconds to 9.2 seconds (concentration dependent, P<0.001 vs. rivaroxaban, n=5); ceiling effect at 0.7 U/mL in all rivaroxaban concentrations rfviia: 5-50 μg/ml significantly shortened PT from 12.9 seconds to minimum of 7.3 seconds (P<0.001 vs rivaroxaban, n=7); all rfviia concentrations significantly reversed rivaroxaban induced PT prolongation by 47-54% (dose dependent) PT Reversal effect rfviia>apcc>pcc Clotting time (CT) PCC: did not affect CT (n=2-3) apcc: reversed CT by 24-40% but only statistically significant at 1656 ng/ml rivaroxaban (P<0.001 vs. rivaroxaban, n=5) rfviia: reduced CT prolongation by 37-53%; significant at 1807 ng/ml rivaroxaban (P<0.001 vs. rivaroxaban, n=4) Lag time PCC: slightly reversed lag time, significant in 1000 ng/ml rivaroxaban at 0.2 U/mL (P<0.001 vs rivaroxaban, n=7) and at higher concentrations of PCC in 500 ng/ml rivaroxaban apcc: significantly reversed lag time for all apcc and rivaroxaban concentrations (P<0.001 vs rivaroxaban, n=5) rfviia: significantly reversed lag time for all apcc and rivaroxaban concentrations (P<0.001 vs rivaroxabn, n=6-8) Lag time reversal: rfviia>apcc>pcc Endogenous thrombin potential (ETP) PCC: significantly reversed rivaroxaban 200 ng/ml at 0.2 U/mL (P<0.01 vs rivaroxaban); increased above baseline at higher PCC concentrations (n=7); slightly increased ETP at higher rivaroxaban concentrations apcc: significantly increased ETP above baseline in 200 ng/ml rivaroxaban and significantly reversed rivaroxaban 500 ng/ml at 0.4 U/mL (P<0.01 vs rivaroxaban, n=5); slight increase of ETP in rivaroxaban 1000 ng/ml at 0.4 U/mL and above (P<0.01 vs. rivaroxaban) Hinds 9

Results (Continued) Authors Conclusions Comments Table 8 (Continued): Perzborn E, Heitmeier S, Laux V, Buchmuller A. 33 rfviia: no significant reversal effect (n=7) Maximum concentration of thrombin generation (C max) PCC: no significant reversal of rivaroxaban (n=7) apcc: significant reversal of rivaroxaban 200 ng/ml for doses above 0.2 U/mL apcc (P<0.05 vs rivaroxaban) and rivaroxaban 500 ng/ml at 1 U/mL apcc (P< 0.05 vs rivaroxaban, n=5) rfviia: significantly increased C max of rivaroxaban 200 ng/ml for all doses of rfviia; significantly increased C max of rivaroxaban 500 ng/ml at doses above 5 μg/ml rfviia (P<0.01 vs. rivaroxaban) and rivaroxaban 1000 ng/ml at 50 μg/ml rfviia (P<0.01 vs rivaroxaban, n=6-8) apcc and rfviia partially reversed inhibition of C max All agents were partially effective for reversal of rivaroxaban-induced anticoagulation rfviia and apcc were more effective than PCC in reversal of PT, CT, lag time of TG Strengths Compared multiple reversal agents at different doses Simulated therapeutic and overdose concentrations of rivaroxaban Limitations Clot based assays may not predict reversal potential of agents In vitro study Agent availability and application in the U.S. Table 9: Escolar G, Fernandez-Gallego, Arellano-Rodrigo E, et al. Reversal of apixaban induced alterations in hemostasis by different coagulation factor concentrates: significance of studies in vitro with circulating human blood. Plos ONE. 2013;8(11):e78696. 34 Purpose To investigate the effects of apixaban on platelets and hemostasis in humans and determine the effectiveness of different factor concentrations at reversing apixaban s effects on hemostasis Design In vitro study design Inclusion Healthy volunteers Exclusion Patients using acetylsalicylic acid, non-steroidal anti-inflammatory or antiplatelet drugs Outcomes within the past 7 days Clotting assays, measure TG, clot formation time (CFT) and maximum clot firmness (MCF) as well as thrombin peak, lag phase, and time peak to determine reversal of apixaban and determine effects in damaged vasculature (see Appendix D, pg 20) Methods Blood samples were donated and apixaban was added to the samples at a concentration of 200 ng/ml, twice the average C max achieved in patients receiving dosages for atrial fibrillation Statistics The following coagulation factor concentrates were tested on apixaban: o rfviia (Novoseven) 270 μg/kg o apcc (FEIBA) 75 U/kg o 4F-PCC (Beriplex) 50 IU/kg (similar to 4F-PCC Kcentra) Assumed a patient s weight was ~70kg Used whole blood samples to analyze clot formation for 45 minutes Coagulation factor concentrates were tested in blood samples and reversal of anticoagulant effect was analyzed via clotting assays Blood was perfused through rabbit aortas to analyze changes when exposed to damaged vascular components ANOVA and Wilcoxon-Mann-Whitney test for Gaussian distribution Hinds 10

Results Authors Conclusions Comments Table 9 (Continued): Escolar G, Fernandez-Gallego, Arellano-Rodrigo E, et al. 34 N=12, ages 22-42 years Table 9a: Thrombin Generation Initiated in Plasma with Phospholipids and Tissue Factor RCL Thrombin CT (s) CFT (s) MCF reagent peak (nm) (mm) Lag phase (min) CON 15.5± 2.3 Apix 34.3± 5.5* Time peak (min) 349.7±30 24.2± 4.1 75.8±23* 42.2± 8.9 165.6± 19.7 357.9± 52.8* 262.9±39.4 53±2.2 923.8± 353.9 40.2±7.7 Apix+ rfviia 15.4± 1.9** 159.8± 41.1# 34.2± 6.6 95.1± 15.9** 140.1± 33.9# 62.3± 2.1# Apix+ apcc 14.2± 1.1** 221.1± 50.1# 37.1±5 102± 9.8** 116.1± 12.5# 62.7± 0.8# Apix+ PCC 25.0± 4.5 169.4±7 42.8± 8.7 326± 86.3 430.7± 117.6 56.9± 1.6# *P<0.01 vs control; **P<0.01 vs apixaban; #P<0.05 vs apixaban; CON:control; Apix:apixaban rfviia and apcc significantly normalized the prolongation to reach peak TG rfviia and apcc significantly reversed lag phase and thrombin peak rfviia and apccs significantly reduced prolongation of CT and CFT Apixaban: apixaban reduced fibrin formation/platelet interaction in damaged vessels o apccs, PCCs, and rfviia returned fibrin levels to baseline rfviia partially reversed apixaban s effects on platelet interactions apcc shows a potential for apixaban reversal apcc may be a first line reversal agent for apixaban Do not recommend rfviia due to thrombogenicity, especially with off label use Results may vary based on specific assays Strengths Examined apixaban concentrations with twice the average C max in patients on therapeutic doses of apixaban Limitations In vitro design limits applicability of study conclusions Short time period for collecting data and availability of agents Rabbit aorta used to study damaged tissue may lead to different conclusions Table 10: Marlu R, Hodaj E, Paris A, Albaladejo P, Crackowski JL, Pernod G. Effect of non-specific reversal agent on anticoagulant activity of dabigatran and rivaroxaban. Thromb Haemostasis. 2012;108:217-224. 35 Purpose To determine the potential of PCC, apcc, and rfviia to reverse the anticoagulant activity of dabigatran and rivaroxaban in human blood Design Randomized, placebo-controlled, crossover, ex-vivo design Inclusion Healthy males, ages 18-45, BMI 18-30 kg/m 2 Exclusion Personal or family history of thrombosis or bleeding disorders, renal or liver impairment Outcomes Clotting assays, measure ETP, thrombin peak, lag time, time to reach maximum peak (see Appendix D, pg 20) Methods Randomized participants received one dose of dabigatran 150 mg or rivaroxban 20 mg 15 day washout period between crossover doses Blood samples collected before then 2 hours after drug administration Reversal agents tested o 4F-PCC (Kanokad) 0.25 U/mL, 0.5 U/mL (25 IU/kg), 1 U/mL (similar to Kcentra) o apcc (FEIBA) 0.25 U/mL, 0.5 U/mL, 1 U/mL (80 IU/kg), 2 U/mL o rfviia (Novoseven) 0.5 μg/ml, 1.5 μg/ml, 3 μg/ml ( 120 μg/kg) Hinds 11

Table 10 (Continued): Marlu R, Hodaj E, Paris A, Albaladejo P, Crackowski JL, Pernod G. 35 Statistics Paired t-test, Shapiro-Wilk s test, Wilcoxon signed-ranks test for normality; Spearman s coefficient of rank correlation for correlation between parameters of TG Results N=10 Rivaroxaban Authors Conclusions Comments ETP PCC: dose dependent increase in ETP (37% with 0.25 U/mL PCC); only dose that reversed ETP to near baseline; overcorrection by higher concentrations apcc: dose dependent increase in ETP (50% increase for 0.25 U/mL p<0.001, only dose that reversed it to near baseline); overcorrection by higher concentrations rfviia: no correction of ETP Thrombin peak PCC: dose dependent correction apcc: dose dependent correction; 1 and 2 U/mL corrected peak close to baseline (P=0.02) rfviia: reduced peak (P=0.02) Lag time PCC: small reduction in lag time (15%) apcc: significant reduction (47%, P<0.0001, rate of thrombin increased to >25 nm/min but did not reach baseline rate) rfviia: complete reversal of lag time to baseline (P<0.001, rate of thrombin increased to >25 nm/min but did not reach baseline rate) Time to reach maximum concentration of thrombin PCC: no effect apcc: decreased time to peak concentration by 30% (p<0.001); increased rate of thrombin formation but did not reach baseline rfviia: decreased time to peak concentration by 44% (P<0.001); increased rate of thrombin formation but did not reach baseline Dabigatran ETP PCC: dose dependent increase in ETP; reached baseline at lowest dose (P=0.02) apcc: dose dependent increase in ETP; reached baseline at lowest dose (P=0.02) rfviia: no correction of ETP Thrombin peak PCC: increased thrombin peak above baseline at lowest dose (P<0.001) apcc: increased thrombin peak above baseline at lowest dose (P<0.001) rfviia: did not increase thrombin peak above baseline Lag time PCC: no significant correction of lag time apcc: all doses except 0.25 U/mL reduced lag time (P<0.001) rfviia: highest dose significantly decreased lag time (P<0.001) Time to reach maximum concentration of thrombin PCC: no effect apcc: significantly reduced by lowest dose of apcc (P=0.03) close to baseline rfviia: reduced time to reach maximum concentration apcc combines the reversal effects of PCC and rfviia to correct both lag time and peak apcc is the most reasonable theoretical option for reversal of rivaroxaban Animal models do not provide evidence for use of rfviia apcc may be useful for dabigatran dosing, but the dose needs to be determined Strengths Randomized, placebo controlled trial Limitations Ex vivo study design limits applicability of study conclusions Duration or response not assessed (short half-lives of reversal agents) Assessed one dose of anticoagulant; availability of agent; no active bleeding Hinds 12

Table 11: Eerenberg ES, Kamphuisen PW, Sijpkens MK, Meijers JC, Buller HR, Levi M. Reversal of rivaroxaban and dabigatran by prothrombin complex concentrate. Circ. 2011;124:1573-1579. 18 Purpose To determine if PCC would reverse the anticoagulant effects of rivaroxaban and dabigatran Design Randomized, double-blind, placebo-controlled, crossover study (blinded to placebo/pcc) Background PCC (Cofact): non-activated 4F-PCC (factors II, VII, IX, X, proteins C and S, antithrombin); similar concentration to Kcentra Inclusion Healthy male individuals (normal blood count, kidney, and liver function, negative for Hepatitis B and C and HIV) Outcomes Rivaroxaban: PT and ETP (see Appendix D, pg. 20); Dabigatran: activated partial thromboplastin time (aptt), ETP, lag time, thrombin time (TT), ecarin clotting time (ECT) (see Appendix D, pg. 20) Methods Received dabigatran (150 mg BID) or rivaroxaban (20 mg BID) for 2.5 days Received either PCC infusion (Cofact) 50 IU/kg or saline on day 3, and monitored blood for 24 hours 11 day washout period Blood collected at baseline, day 3, 15 and 30 minutes, then 1, 2, 4, 6, and 24 hours after infusion Statistics Paired t-test and repeated measures using ANOVA for confirmation; Between group comparison used the independent-sample t test Results N=12, Age: 24 ± 4 years, BMI: 23 ± 3 kg/m 2 Table 11a: Eerenberg Results Parameter Baseline Rivaroxaban Dabigatran Pre-PCC Post-PCC Pre-PCC Post-PCC Authors Conclusions Comments PT (sec) 12.3±0.7 15.8±1.3 (P<0.001) ETP (%) 92±22 51±21 (P=0.002) 12.8±1.0 (P<0.001) 114±26 (P<0.001) aptt (sec) 33.6±3.3 -- -- 59.4±15.8 (P<0.001) ETP lag 2.9±0.4 -- -- 7.5±2.5 time (min) (P<0.001) ECT (sec) 33±1 -- -- 69±26 (P=0.002) -- -- -- -- 70.3±10.3 (P=0.64) 8.7±2.6 (P=0.20) 86 ±20 (P=0.08) Studied effects persisted until the last measurement at 24 hours unless specified below TT prolongation remained immeasurable for at least 6 hours after PCC infusion 4F-PCC significantly increased ETP (amount of thrombin generated) in rivaroxaban samples and significantly reversed PT prolongation 4F-PCC did not significantly reverse aptt, ETP, or ECT in dabigatran samples Saline did not significantly affect results after the anticoagulant was administered Adverse effects: small hematoma at infusion site occurred in 2 rivaroxaban and 2 apixaban patients, gingival bleeding in 2 dabigatran patients Nonactivated 4F-PCC reversed the effect (PT, ETP) of full-dose rivaroxaban immediately but did not reverse dabigatran at the dose studied No support to use nonactivated 4F-PCC for reversal of direct thrombin inhibitors Strengths Randomized, double-blind, placebo controlled trial Anticoagulants and 4F-PCC administered to participants Limitations Participants received financial compensation All male participants Rivaroxaban dose higher than normal treatment dose Accuracy of coagulation tests in monitoring reversal potential of 4F-PCC No active bleeding to assess actual reversal Used only one dose of one PCC agent; availability of agent Hinds 13

VI. A. Levi et al conducted a trial examining the reversal of rivaroxaban using 3F-PCC (Profilnine SD) and 4F-PCC (Beriplex) 35 i. Randomized, open-label single center, parallel group study, n=35 1. Inclusion criteria: healthy, 18-55 years old, normal creatinine clearance, BMI 18-30 kg/m 2, not pregnant, normal coagulation tests at baseline 2. Exclusion criteria: history of thrombosis, thrombophilia/bleeding tendency/coagulopathy ii. Methods: Received rivaroxaban 20 mg twice daily for 4.5 days, then given one dose of either 50 IU/kg 3F-PCC (n=12), 50 IU/kg 4F-PCC (n=10), or saline bolus (n=12) 4 hours after the last rivaroxaban dose 1. Blood samples collected pre-dose daily and up to 28 hours after last rivaroxaban dose iii. Outcomes: PT, thrombin generation assay, aptt, anti Xa level Table 12: Levi et al Reversal Results of 3F-PCC and 4F-PCC on Rivaroxaban 36 Rivaroxaban 3F-PCC 4F-PCC ETP Decreased Increased faster than 4F-PCC to above pre-study levels Increased above pre-study levels Lag time Prolonged (2-3 min) No effect No effect PT Prolonged (21 sec) Reduced (20sec)* Reduced (17.5 sec)* Thrombin Decreased Increased Increased < 3F-PCC level (slow response initially) Time to peak Prolonged Decreased Decreased < 3F-PCC aptt Prolonged (8 sec) Prolonged then return to baseline (4 sec) Prolonged then return to baseline (6 sec) Anti-Xa Increased No effect No effect *30 minutes after PCC administration iv. Adverse effects: 18 patients (51.4%) 1. 15 reported during rivaroxaban monotherapy, 2 in 3F-PCC, 2 in 4F-PCC 2. Most common adverse effects: abdominal discomfort, gingival bleeding, back pain, headache (8.6% for each category) a. Gingival bleeding (n=3), mouth hemorrhage (n=1) b. No thromboembolic events reported v. Conclusion: 4F-PCC and 3F-PCC partially reverse anticoagulant effects of rivaroxaban and appear safe and well tolerated 1. 3F-PCC and 4F-PCC had different intensity of reversal on PT and thrombin generation Summary of evidence for reversal of new oral anticoagulants A. Human studies i. Apixaban 1. apccs and rfviia partially reverse its anticoagulant parameters 34 2. 4F-PCCs did not reverse anticoagulant parameters ii. Dabigatran 1. 4F-PCC reversal of dabigatan conflicting a. Activated 4F-PCC reversed peak thrombin and lag time 34 b. Non-activated 4F-PCC did not reverse aptt, ETP ECT 32 2. rfviia reverses lag time iii. Rivaroxaban 1. apcc and rfviia are more effective than 4F-PCC for reversal of effects 33 2. 4F-PCC partially reverses rivaroxaban Hinds 14

VII. B. Murine Studies (see Appendix C, pg 20) 37 i. Dabigatran 1. apcc decreased mortality while rfviia did not 2. rfviia may have less reversal of hemostasis compared to apcc C. Rabbit (see Appendix C, pg 20) 37 i. Apixaban 1. rfviia reduced bleeding time and PCC did not ii. Dabigatran 1. apcc decreased blood loss iii. Rivaroxaban 1. rfviia and PCC reduced bleeding time but not blood loss Recommendations for the reversal of new oral anticoagulants A. Recommendations for reversal of NOACs i. Consider additional reversal methods of NOACs in severe bleeding defined as a decrease in Hb 2 g/dl and/or transfusion of 2 units of whole blood 1. Apixaban a. Consider administration of apcc in healthy adults b. If not on formulary, 4F-PCC may be considered 2. Dabigatran a. Consider administration of apcc in healthy adults b. If all supportive measures fail (including hemodialysis), may consider administration of apcc in healthy adults c. If apcc not on forumulary, 4F-PCC may be considered 3. Rivaroxaban a. Consider administration of apcc in healthy adults b. If not on formulary, 4F-PCC may be considered ii. Use of these agents can increase risk of thrombosis iii. Appropriate stratification and consent of patient is warranted prior to use of 4F- PCCs 1. Only use if bleeding is severe and immediate action is required 2. Evidence supporting a specific dose is lacking 3. Limit repeat doses due to thrombosis risk 4. If bleeding not severe, wait for systemic drug concentration to decrease since agents have short half-life iv. Randomized controlled trials needed to determine most appropriate reversal strategies and dose in the setting of acute bleeding, overdoses, comborbid conditions, and renal impairment 1. Studies needed to determine if reversal of anticoagulant monitoring parameters by PCCs/clotting factors correlates with reversal of bleeding v. New drugs on the horizon 1. Established reversal agents coming soon Hinds 15

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26. NovoSeven RT Coagulation Factor VIIa (recombinant) [package insert]. Bagsvaerd, Denmark; Novo Nordisk; Revised July, 2014. http://www.novo-pi.com/novosevenrt.pdf. Accessed September 20, 2014. 27. Portola Pharmaceuticals. Portola pharmaceuticals announces first phase 2 results demonstrating extended duration infusion with andexanet alfa (PRT4445). http://www.portola.com/clinicaldevelopment/andexanet-alfa-prt4445-fxa-inhibitor-antidote/. Updated 2014. Accessed September 25, 2014. 28. Cother MA, Kitt M, McClure M, et al. Randomized, double-blind, placebo-controlled single ascending dose pharmacokinetic and pharmacodynamics study of PRT064445, a universal antidote for factor Xa inhibitors (abstract). Arterioscler Thromb Vasc Biol. 2013;3:A10. 29. Lauw MN, Cppens M, Eikelboom JW. Recent advances in antidotes for direct oral anticoagulants: their arrival is imminent. Can J Cardiol. 2014;30:381-384. 30. Crowther MA, Marthur V, Kitt M, et al. A phase 2 randomized, double-blind, placebo-controlled trial demonstrating reversal of rivaroxaban-induced anticoagulation in healthy subjects by andexanet alfa (PRT064445), an antidote for FXa inhibitors (abstract). Blood. 2013;122:A3636. 31. Laulicht B, Bakhru S, Lee C, et al. Abstract 11395: small molecule antidote for anticoagulants (abstract). Circ. 2012;126:A11395. 32. Schneider G. An antidote on the horizon? An update on the progress toward achieving reversibility for the new oral anticoagulants. Clinical Correlations. http://www.clinicalcorrelations.org/?p=7373. Updated February, 2014. Accessed September 25, 2014. 33. Perzborn E, Heitmeier S, Laux V, Buchmuller A. Reversal of rivaroxaban-induced anticoagulation with prothrombin complex concentrate, activated prothrombin complex concentrate and recombinant activated factor VII in vitro. Thromb Res. 2014;133:671-681. 34. Escolar G, Fernandez-Gallego, Arellano-Rodrigo E, et al. Reversal of apixaban induced alterations in hemostasis by different coagulation factor concentrates: significance of studies in vitro with circulating human blood. Plos ONE. 2013;8(11):e78696. 35. Marlu R, Hodaj E, Paris A, Albaladejo P, Crackowski JL, Pernod G. Effect of non-specific reversal agent on anticoagulant activity of dabigatran and rivaroxaban. Thromb Haemostasis. 2012;108:217-224. 36. Levi M, Moore KT, Castillejos CF, et al. Comparison of three-factor and four-factor prothrombin complex concentrates regarding reversal of the anticoagulant effects of rivaroxaban in healthy volunteers. J Thromb Haemost. 2014;12(9):1428-1436. 37. Nutescu EA. Oral anticoagulant therapies: balancing the risks. Am J Health Syst Pharm. 2013;70;S3- S11. 38. Baxter Healthcare Corportation. About FEIBA NF. http://www.feiba.com/us/hcp/hcp_aboutus.html. Updated 2014. Accessed September 25, 2014. 39. Lee FM, Chan, AK, Lau kk, Chan HH. Reversal of new, factor-specific oral anticoagulants by rfviia, prothrombin complex concentrate, and activated prothrombin complex concentrate: a review of animal and human studies. Thromb Res. 2014;133:705-713. 40. Khoo TL, Weatherburn C, Kershawy G, Reddel CJ, Curnow J, Dunkley S. The use of FEIBA in the correction of coagulation abnormalities induced by dabigatran. Int J Lab Hematol. 2013;35:222-224. Hinds 17

Appendix A Mechanism of action Side effects Table 13: New Oral Anticoagulants Characteristics 7,8,9 Dabigatran Apixaban Rivaroxaban Reversibly Inhibits free Inhibits free inhibits factor Xa and factor Xa and thrombin clot-bound prothrombinase (factor IIa) factor Xa and activity prothrombinase activity Bleeding (2.7%/yr dabigatran vs. 3.36%/yr warfarin for major bleeding*) gastrointestinal (35% vs. 24% warfarin) Bleeding (1.1% in knee/hip replacement) nausea (2.6% in knee/hip replacement) Bleeding (1% in major bleeding*) dyspepsia (1.3% vs. 0.7% placebo) upper abdominal pain (1.7% vs 0.2% placebo) * Major bleeding: hemoglobin decreased by 20 gm/liter, transfusion 2 units blood, symptomatic bleeding in a critical area/organ 9,11 Table 14: Dabigatran Dosage Adjustments 7 Creatinine Clearance (CrCl) Dose >30 ml/min 150 mg twice daily 15-30 ml/min 75 mg twice daily 30-50 ml/min + P-GP inh* <30 ml/min + P-GP inh <15 ml/min or dialysis 75 mg twice daily Avoid use No recommendation *May decrease dosage to 75 mg twice daily with the following; P-GP inh: dronedarone or ketoconazole Table 15: Apixaban Dosage Adjustments 8 Indication Dose Dosage Adjustment/Duration Nonvalvular atrial fibrillation 5 mg twice daily 2.5 mg twice daily if at least 2: age 80, body weight 60 kg, serum creatinine 1.5 mg/dl DVT prophylaxis post hip/knee replacement surgery and reduction in risk of recurrent DVT/PE Treatment of DVT 2.5 mg twice daily Hip surgery: 35 days Knee surgery: 12 days DVT/PE: at least 6 months 10 mg twice daily, then 10 mg x 7 days, 5 mg thereafter 5 mg twice daily Table 16: Rivaroxaban Dosage Adjustments 9 Indication Dose Dosage Adjustment/Duration Nonvalvular atrial fibrillation/reduce recurrence of 20 mg daily CrCl 15-50 ml/min: 15 mg daily CrCl<15 ml/min: avoid use DVT/PE Treatment of DVT/PE 15 mg twice daily x 21 days then 20 mg daily CrCl<30 ml/min: avoid use Prophylaxis of DVT following hip or knee replacement surgery 10 mg daily CrCl<30 ml/min: avoid use Hip surgery: 35 days duration Knee surgery: 12 days duration Hinds 18

Appendix B Figure 2: apcc (FEIBA) Mechanism of Action 38 Appendix C Author/ Date Zhou 2011 Lambourne 2012 Zhou 2013 Perzborn 2013 Pragst 2012 Godier 2012 Godier 2013 Perzborn 2013 Khoo 2013 Table 17: Summary of NOAC Reversal Trials (human and non-human studies) 39,40 Model NOAC Reversal Agent murine dabigatran 4F-PCC rfviia murine dabigatran rfviia PCC rfviia+pcc apcc murine dabigatran Murine FFP Human rfviia rat rivaroxaban rfviia PCC apcc Conclusion 4F-PCC decreased mortality to baseline, prevented growth of hematoma; rfviia did not All failed to reduce blood loss; apcc and rfviia+pcc reduced bleeding time; rfviia may have less hemostatic reversal potential relative to apcc All reversal agents significantly reduced hematoma expansion; rfviia reversed hematoma expansion and improved neurological deficits All reduced prolongation of bleeding time rabbit dabigatran 4F-PCC Reduced blood loss and decreased time of hemostasis in a dose-dependent manner rabbit rivaroxaban rfviia PCC rabbit apixaban (IV) rfviia PCC baboon rivaroxaban rfviia PCC apcc Both significantly reduced bleeding time but not reversal of blood loss Neither significantly decreased blood loss; rfviia partially reduced bleeding time apcc normalized bleeding time; rfviia partially decreased bleeding time human dabigatran apcc apcc normalized peak thrombin generation/lag time Hinds 19

Appendix D Table 18: Laboratory Assays for Assessment of NOACs 35,37 Parameter Definition Application Time from the addition of calcium to formation of a fibrin clot Activated partial thromboplastin time (aptt) Anti-Xa Clot formation time (CFT) Clotting time (CT) Ecarin clotting time (ECT) Endogenous thrombin potential (ETP) Lag time (LT) Maximum clot firmness (CFT) Maximum concentration of thrombin (C max) Prothrombin time (PT) Measures the change in absorbance of specific clotting factor Xa activity Speed of clot formation from 2 mm to 20 mm above baseline in seconds Measures conversion of fibrinogen to fibrin (time until clot reaches 2 mm) Measures thrombin generation (ecarin activates prothrombin) Area under the curve, maximum amount of thrombin produced Time to start thrombin generation Firmness of clot formed, strength of fibrin/platelet clot Maximum concentration of thrombin generated Linear response to dabigatran up to therapeutic dose (not quantitative in overdose) Monitoring factor Xa inhibitors (if calibration and appropriate control agent available) Prolonged by apixaban May be useful for dabigatran to steady-state levels; increased by rivaroxaban and apixaban Most useful test for monitoring dabigatran; dose dependent increase when dabigatran present Decreased by rivaroxaban and dabigatran Prolonged by dabigatran, rivaroxaban, apixaban Decreased by apixaban Decreased by rivaroxaban Time for plasma to clot after calcium and thromboplastin added Prolonged when dabigatran present (not as extensive as aptt response); more responsive to factor Xa inhibitors Thrombin time (TT) Activity of thrombin in plasma Less applicable than ECT, increased when dabigatran present (qualitative) Prolonged by apixaban, rivaroxaban, dabigatran Time to peak Time to reach maximal effect of thrombin generation Other labs Complete blood count, Hemoglobin/hematocrit and platelets serum creatinine (bleeding), creatinine clearance (renal function) Coagulation assays and effect on patient outcomes not established; different reagents used in laboratory settings have different responsiveness Hinds 20