Anticoagulation with the Novel, Small-molecule Factor XIa (fXIa) Antagonist, EP-7041, Prevents # PB0166 Oxygenator Clotting but Conserves Hemostasis in a Canine Extracorporeal Circulation (ECMO) Model M.A. Kurz1, C.V. Pollack, Jr.1, J.M. Connors2, J.H. Levy3, C. Drummond4, D. Klein5, H.D. Josuns5, R. Pielemeier4, J. Buchino4, P. Osborne4, N.J. Hayward1 1eXIthera Pharmaceuticals, Inc., Westborough, USA, 2Brigham and Women's Hospital, Hematology, Boston, USA, 3Duke Clinical Research Institute, Anesthesiology, Durham, USA, 4Charles River Labs, Surgical Services, Mattawan, USA, 5Borgess Health/Comprehensive Care Services, Kalamazoo, USA

BACKGROUND / AIMS KEY FINDINGS RESULTS Figure 1a Pressure Proximal to Membrane Oxygenator During Low-flow ECMO Figure 1b Bleeding During Low-flow ECMO Bleeding and thrombosis are the most common complications of veno-venous (V-V) ECMO 350 The boluses and infusions of EP-7041 and rapidly achieved target aPTTs (Fig 3). Despite Vehicle (n=3) 1.2 maintenance of steady-state aPTT near 1.5× BL, the rate of hemorrhage increased over the support for patients with respiratory failure. ECMO requires anticoagulation to prevent Heparin (n=8) *P <0.05 300 vs Heparin course of the ECMO run in heparin-treated animals, but not in EP-7041-treated animals (Fig 4). clotting in the circuit, but heparin and other thrombin inhibitors significantly increase EP-7041 Dose 1 (n=6) 1.0 EP-7041 Dose 2 (n=5) Fibrinogen levels decreased in all groups while on ECMO. There were no significant changes in PT bleeding risk at therapeutic concentrations. 250 0.8 with either heparin or EP-7041 (Fig 5). The bolus dose of EP-7041 overshot steady-state, but Inhibition of coagulation Factor XI/XIa is an appealing pathway for antithrombotic support of 200 plasma concentrations stabilized prior to ECMO initiation (Fig 6). 0.6 ECMO. Selective inhibition of the contact pathway of coagulation could improve bleeding Figure 3 Figure 5 150 aPTT in Hounds Undergoing ECMO PT and Fibrinogen in Hounds Undergoing ECMO risk, and because FXI is linked with the inflammatory and complement systems, it can also 2.5 2.5

0.4 Heparin PT (n=12) Dose 1 PT (n=10) Oxygenator Pressure (mm/hg) Pressure Oxygenator be viewed as a biologically plausible target for the prevention of pathologic thrombosis - 100 Dose 2 PT (n=9) Vehicle PT (n=3)

Pre * 2.0 2.0 Hep-Fibrinogen Dose 1 Fibrinogen during ECMO. 0.2 BL) from

50 P=0.07 * Dose 2 Fibrinogen Vehicle Fibrinogen

Δ Loss during ECMO (g/min) ECMO during Loss Blood

EP-7041 is a parenteral, potent, and selective, small-molecule FXIa inhibitor with Fold

- from BL) from

0 0.0 1.5 × 1.5 Δ

pharmacodynamic and pharmacokinetic characteristics that appear well-suited for use in a 0 30 60 90 120 150 Vehicle Heparin EP-7041 EP-7041 Fold Fold

Time after starting ECMO Dose 1 Dose 2 - critical care environment. It is primarily excreted through the . × 1.0 1.0

Aims: We aimed to show that intravenous administration of EP-7041 prevents clotting in the aPTT( As expected, “low-flow” conditions near the MO’s labeled lower limit of 500 mL/min Vehicle (n=3) Heparin (n=12) EP-7041 Dose 1 (n=10) EP-7041 Dose 2 (n=9) ECMO membrane oxygenator while causing less bleeding than heparin. ( Fibrinogen or PT accelerated thrombotic complications of ECMO. Pressure increased in all groups during 0.5 0.5 -30 0 30 60 90 120 150 -30 0 30 60 90 120 150 ECMO. All Vehicle controls reached the system limit of 350 mmHg, requiring termination Time after starting ECMO Time after starting ECMO Figure 4 Timing of Bleeding in Hounds Undergoing ECMO Figure 6 Plasma Concentration of EP-7041 MATERIALS & METHODS within 1 hour (Fig 1a). One Group 1 EP-7041 animal also occluded, but all other treated 1.8 10000 Vehicle (n=3) animals had only minor or transient pressure increases. Despite maintaining steady-state 1.6 Heparin (n=12) The canine model of V-V ECMO employs standard tubing, pumps, and oxygenator 1.4 EP-7041 Dose 1 (n=10) aPTT with heparin dosing near 1.5× BL, bleeding rates and total measured blood loss EP-7041 Dose 2 (n=9) components (Medtronic) in large (>24kg) hounds. Animals were anesthetized and 14F 1.2 during ECMO were more than 5× greater with heparin treatment vs. EP-7041-treatment *P <0.05 cannulae were placed via cutdown in the jugular and femoral veins for venous drainage and 1.0 vs Heparin (Figs. 1b, 4, p <0.05). 1000 return. Heparin and EP-7041 were dosed to target aPTT of 1.5× - 2× baseline (BL); diluent 0.8

(normal saline) was administered to match active agent fluid volume as a negative (Vehicle) 0.6 (ng/mL) [7041]p

Figure 2a Figure 2b Bleeding During Standard-flow ECMO (g/min) Bleeding Pressure Proximal to Membrane Oxygenator During Standard-flow ECMO 0.4 * control. Study drug boluses and infusions were started 30 min prior to initiation of ECMO to * EP-7041 Dose 1 (n=10) 200 1.2 * * 0.2 * * EP-7041 Dose 2 (n=5) allow anticoagulation to reach steady-state. *P <0.05 * * vs Heparin 0.0 100 Low-flow (20 mL/kg/min) or Standard-flow (60 mL/kg/min) ECMO circulation was 1.0 0-15 30-45 60-75 90-105 120-135 -30 0 30 60 90 120 150 Time after starting ECMO (min) Time after starting ECMO (min) maintained for 2.5 hours or until pressure proximal to the oxygenator reached 350 mmHg 150 0.8 due to clotting. Bleeding rates were determined by placing preweighed sponges on incision SUMMARY/CONCLUSION sites for 15 minutes at 30-minute intervals. 100 0.6 In this canine ECMO model, heparin dosed to an aPTT of 1.5× BL prevented

0.4 Oxygenator Pressure (mm/hg) Pressure Oxygenator Treatment Group Treatment Goal Dose N - occlusion of the oxygenator or circuit, but consistently caused cutaneous bleeding 50 Heparin (n=4) * Pre which increased as ECMO progressed. Vehicle Control “Normal” coagulation Saline 1 mL/kg bolus + 3 EP-7041 Dose 1 (n=4) 0.2

5 mL/kg/hr infusion EP-7041 Dose 2 (n=4) * EP-7041 prevented increases in oxygenator resistance as effectively as heparin Blood Loss during ECMO (g/min) ECMO during Loss Blood Steady-state aPTT = 1.5×-2.0× BL 25 IU/kg IV Bolus + 12 0 0.0 Heparin Control 0 30 60 90 120 150 Heparin EP-7041 EP-7041 but, unlike heparin, did not significantly impede hemostasis. Our findings support 20-22 IU/kg/hr IV Infusion Time after starting ECMO Dose 1 Dose 2 the key role of Factor XIa in driving thrombosis – but not hemostasis – during EP-7041 Dose 1 Steady-state aPTT = 1.5×-2.0× BL 0.3 mg/kg IV Bolus + 10 ECMO. As a Factor XIa antagonist EP-7041 is: 0.6 mg/kg/hr IV Infusion With “standard flow,” pressure was significantly higher at ECMO start, but only gradually • Potent and highly selective EP-7041 Dose 2 Steady-state aPTT = 2.0×-2.5× BL 1.0 mg/kg IV Bolus + 9 increased during ECMO (Fig 2a). There were no differences in pressure between 2.2 mg/kg/hr IV Infusion treatment groups under standard-flow conditions, but similarly to low-flow groups, • Short-acting ▪ Solutions prepared to deliver similar total intravenous infusion volumes to all treatment groups (1 mL/kg bolus + 5 heparin-treated animals consistently experienced significantly more bleeding (Fig 2b). • Predictable PK/PD mL/kg/hr infusion) There were no differences in study-drug concentrations or measured hematologic Further study of EP-7041 is warranted in ECMO, in other intravascular ▪ ECMO circuit and catheter flush solutions contained study drug at concentrations expected in plasma at steady-state variables between low- and standard-flow groups. instrumentation, and in prevention and treatment of thrombosis in other settings.