Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor 1, the imidazole linker was first replaced with a pyrazole moiety to establish a polar C–H···water hydrogen-bonding interaction. Then, structure-based drug design was employed to modify lead molecule 2d in the P1′ and P2′ regions, with substituents interacting with key residues through various nonclassical interactions. As a result, a potent FXIa inhibitor 3f (K i = 0.17 nM) was discovered. This compound demonstrated oral bioavailability in preclinical species (rat 36.4%, dog 80.5%, and monkey 43.0%) and displayed a dose-dependent antithrombotic effect in a rabbit arteriovenous shunt model of thrombosis.

JNJ-64179375 (JNJ-9375) is a recombinant human IgG4 monoclonal antibody engineered to mimic an IgA antibody that was identified in a patient who exhibited markedly prolonged clotting times but without spontaneous bleeding episodes over several years of follow-up. The crystal structure of the JNJ-9375 antigen-binding fragment/thrombin complex showed an almost identical binding mode to that of the patient IgA. In the current study, we characterized the in vitro and in vivo properties of JNJ-9375. Surface plasmon resonance studies demonstrated that JNJ-9375 binds to a-thrombin with high affinity and specificity (K D : 0.8 nM for human thrombin). JNJ-9375 produced concentration-dependent prolongation of in vitro clotting assays in human plasma, including thrombin time (TT), ecarin clotting time, prothrombin time, and activated partial thromboplastin time, with EC 2X values of 4.4, 12.4, 172.6, and 202.7 mg/ml, respectively. JNJ-9375 inhibited thrombin-induced platelet aggregation in human plasma with an IC 50 value of 52.6 nM (7.8 mg/ml) and produced concentration-dependent prolongation of reaction time tested by thromboelastography. JNJ-9375 pretreatment resulted in dose-dependent reduction in thrombus formation in the rat arteriovenous (AV) shunt model of thrombosis. Robust efficacy was observed at 0.3 mg/kg accompanied by 1.5Â of TT. Bleeding was increased at 3 mg/kg in a rat tail transection bleeding model demonstrating a therapeutic index of 10Â compared with 1Â for apixaban in the same models. Our data suggest that thrombin exosite I inhibition is efficacious against thrombosis in a pretreatment prevention animal model. SIGNIFICANCE STATEMENT JNJ-9375 is a novel, fully human monoclonal antibody that binds to the exosite I region of thrombin with high affinity and specificity. JNJ-9375 concentration dependently prolonged clotting times and inhibited thrombin-induced platelet aggregation in in vitro assays based on its mechanism of action. In an in vivo rat AV shunt model, JNJ-9375 prevented thrombus formation in a dose-dependent fashion while demonstrating reduced bleeding risk. The present study demonstrated the antithrombotic effects of inhibiting the exosite I region of thrombin when given in a prevention mode in preclinical animal models.

Background and Purpose Antigen‐binding fragment (Fab) reversal agents were developed to reverse, in bleeding emergency, the long‐acting anticoagulant effect of JNJ‐64179375 (JNJ‐9375), a monoclonal antibody that binds exosite‐1 on thrombin. Experimental Approach The pharmacokinetic and pharmacodynamic (PK/PD) activities of three reversal agents of varying in vitro binding affinities to JNJ‐9375 were characterised in cynomolgus monkeys. The time course of JNJ‐9375 anticoagulant activity and reversal effects of each agent were evaluated. A mechanism‐based PK/PD model, which integrated free serum concentrations of reversal agent, total and free serum concentrations of JNJ‐9375, and thrombin time, was developed to quantitatively relate JNJ‐9375 neutralisation to reversal of induced thrombin time prolongation. Model‐based allometric scale‐up of the lead reversal agent and the PK/PD relationship of JNJ‐9375 in healthy volunteers were utilised to predict clinical dosing regimens. Key Results Lowering of free JNJ‐9375 by the reversal agents corresponded with reversal of thrombin time prolongation. Total JNJ‐9375 displayed typical mAb clearance at 2.75 ml·day−1·kg−1, whereas reversal agents cleared faster between 1400 and 2400 ml·day−1·kg−1. The model‐estimated in vivo KD values for JNJ‐9375 reversal agents were 9 nM (ICHB‐256), 0.4 nM (ICHB‐281) and 13.7 pM (ICHB‐164), in rank‐ordered agreement of their KD values determined in vitro. The three reversal agents exhibited different neutralisation characteristics in vivo, governed primarily by their binding kinetics to JNJ‐9375. The model predicted a priori free JNJ‐9375 kinetics after dosing ICHB‐164 (JNJ‐67842125) and JNJ‐9375 under a different regimen. Conclusion and Implications The results enabled selection of JNJ‐67842125 as the reversal agent for JNJ‐9375.

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