In vitro comparison of eptacog beta and eptacog alfa with antithrombin lowering (simulated fitusiran) using thrombin generation assay

Abstract Introduction: Eptacog beta, an FDA approved bypassing agent for the treatment of bleeds in patients with hemophilia A and B with inhibitors differs from eptacog alfa in its glycosylation profiles and platelet binding properties. Fitusiran, a non-factor therapy that suppresses antithrombin (AT) production was recently approved for prophylaxis in hemophilia A and B, with or without inhibitors. For persons with hemophilia and inhibitors, breakthrough bleeds on fitusiran prophylaxis would require treatment with a bypassing agent. In fitusiran clinic trials, eptacog alfa was used at reduced dosing (45µg/kg) for bleed management. To date, the combined effect of eptacog beta and fitusiran on in vitro thrombin generation (TG) has not been described. Our study aims to characterize the impact of eptacog beta (eB) and AT lowering (to mimic fitusiran) on TG parameters and compare to that of eptacog alfa (eA). Methods: Thrombin generation assay with a tissue factor trigger was used to evaluate peak (nM) and endogenous thrombin potential (ETP (nM*min)) in pooled FVIII deficient plasma spiked with concentrations of eA ranging from 0-2 µg/mL (0-120 µg/kg), or eB ranging from 0-3.22 µg/mL (0-300 µg/kg). AT levels (15 - 92%) were achieved via an anti-human AT antibody. AT activity was measured by a chromogenic assay. Pooled normal plasma (PNP) was used as control. Results: TG parameters of PNP were peak (71.2-154.8) and ETP (879.08 -1472.43). TG parameters that exceeded the upper limit of the assay were noted at the following combinations: 15% AT with > 0.17µg/mL (25 µg/kg) eB; > 0.25µg/mL (15 µg/kg) of eA 20% AT with > 0.45 µg/mL (50 µg/kg) eB; > 0.25 µg/mL (15 µg/kg) eA 25% AT with > 1.28 µg/mL (125 µg/kg) eB; > 0.75 µg/mL (45 µg/kg) eA 30% AT with > 2.44 µg/mL (225 µg/kg) eB; > 1.0 µg/mL (60 µg/kg) eA 35% AT with > 2 µg/mL (120 µg/kg) eA High TG parameters were noted at the following combinations: 20% AT with eB: 0.17µg/mL (25 µg/kg) [peak 174, ETP 3383] and 0.28µg/mL (35 µg/kg) [peak 181, ETP 3567] 25% AT with eB: 0.17µg/mL (25 µg/kg) [peak 164, ETP 3331]; 0.28 µg/mL (35µg/kg) [peak 174, ETP 3461]; 0.45 µg/mL (50 µg/kg) [peak of 177.9, ETP of 3545]; 0.57 µg/mL (75 µg/kg) [peak 177.37, ETP of 3705]; 25% AT with eA: 0.25 µg/mL (15 µg/kg) [peak 176.85, ETP 3357]; 0.5 µg/mL (30 µg/kg) [peak 177.27, ETP 3961]; 30% AT with eB: 0.28 µg/mL (35µg/kg) [peak 165, ETP 3280]; 0.45 µg/mL (50 µg/kg) [peak 166.14, ETP 3417]; 0.57 µg/mL (75 µg/kg) [peak 175.24, ETP 3616]; 1.28 µg/mL (125 µg/kg) [peak 179, ETP 3651]; 1.56 µg/mL (150 µg/kg) [peak 181, of 3715] 30% with eA: 0.25 µg/mL (15 µg/kg) [peak 165, ETP 3265]; 0.5µg/mL (30 µg/kg) [peak 178.34, ETP 3795]; 0.75 µg/mL (45 µg/kg) [peak 179, ETP 4010] 35 % AT with eB: 0.45 µg/mL (50 µg/kg) [peak 158, ETP 3286]; 0.57 µg/mL (75 µg/kg) [peak 159, ETP 3523]; 1.28 µg/mL (125 µg/kg) [peak 168, ETP of 3625]; 1.56 µg/mL (150 µg/kg) [peak 167, ETP 3672]; 2.44 µg/mL (225 µg/kg) [peak 171, ETP 3788]; 3.22 µg/mL (300 µg/kg) [peak 176, ETP 3908] 35% with eA: 0.25 µg/mL (15 µg/kg) [peak 155, ETP of 3173]; 0.5µg/mL (30 µg/kg) [peak 166, ETP 3704]; 0.75 µg/mL (45 µg/kg) [peak 175, ETP 3823]; 1.0 µg/mL (60 µg/kg) [peak 177, ETP 4077]; 1.5 µg/mL (90 µg/kg) [peak 181, ETP 4249] Normal TG were seen at the following combinations: 30% AT with: 0.17 µg/mL (25 µg/kg) eB [peak 153, ETP 3184] 35 % AT with: 0.17 µg/mL (25 µg/kg) eB [peak 146, ETP 3045]; 0.28 µg/mL (35µg/kg) eB [peak 149, ETP 3120] AT levels >40 % with all doses of eA and eB resulted in normal or slightly above normal TG parameters. Conclusions: These results demonstrate that in vitro both eptacog beta and eptacog alfa similarly increase TG when AT levels are reduced to fitusiran target ranges. Eptacog alfa doses of 45µg/kg, used to treat bleeds in patients on fitusiran, had similar TG parameters to the 75µg/kg and 125µg/kg doses of eptacog beta. This data provides in vitro proof of concept supporting the use of eptacog beta for the treatment of breakthrough bleeds of patients on fitusiran prophylaxis. The clinical implications of the amplified in vitro TG remain to be seen, and human in vivo studies are needed to support patient bleed management guidelines.