First-in-human study to assess the safety, pharmacokinetics, and pharmacodynamics of BMS-986141, a novel, reversible, small-molecule, PAR4 agonist in non-Japanese and Japanese healthy participants.

BMS-986141 is a novel, oral, protease-activated, receptor 4 (PAR4)-antagonist that exhibited robust antithrombotic activity and low bleeding risk in preclinical studies. The pharmacokinetic, pharmacodynamic, and tolerability profiles of BMS-986141 in healthy participants were assessed in a randomized, double-blind, placebo-controlled, single-ascending-dose (SAD; N = 60) study; a multiple-ascending-dose (MAD; N = 32) study; and a Japanese MAD (JMAD; N = 32) study. Exposure was dose-proportional for BMS-986141 2.5 mg and 150 mg; maximum concentrations were 17.6 ng/mL and 958 ng/mL; and areas under the curve (AUC) to infinity were 183 h* × ng/mL and 9207 h* × ng/mL, respectively. Mean half-life ranged from 33.7 to 44.7 hours across dose panels. The accumulation index following once-daily administration for 7 days suggested a 1.3- to 2-fold AUC increase at steady state. In the SAD study, BMS-986141 75 and 150 mg produced ≥80% inhibition of 25-100 µM PAR4 agonist peptide (AP)-induced platelet aggregation, without affecting PAR1-AP-induced platelet aggregation, through ≥24 hours postdose. In the MAD and JMAD studies, BMS-986141 doses ≥10 mg completely inhibited 12.5 µM and 25 µM PAR4-AP-induced platelet aggregation through 24 hours. This study found BMS-986141 was safe and well tolerated, with dose-proportional pharmacokinetics and concentration-dependent pharmacodynamics in healthy participants over a wide dose range. ClinicalTrials.gov ID: NCT02341638.

Why was the study done? Antiplatelet therapies have shortcomings that limit their clinical utility, and there is an unmet need for a new, safe, and effective antiplatelet agent with reduced bleeding risk.PAR4 antagonists are a promising novel class of antiplatelet drugs due to late-stage inhibition of thrombus growth with minimal effects on platelet-driven hemostasis.BMS-986141 is a novel, potent, orally bioavailable, small-molecule antagonist specific for PAR4.What is new? BMS-986141 is safe and well tolerated, with dose-proportional pharmacokinetics and concentration-dependent pharmacodynamics in healthy participants over a wide dose range.BMS-986141 has robust antithrombotic activity and low bleeding risk.What is the impact? BMS-986141 has the potential to improve the benefit­risk of antiplatelet therapy in patients with atherothrombosis.

An optimized agonist peptide of protease-activated receptor 4 and its use in a validated platelet-aggregation assay.

Protease-activated receptor 4 (PAR4) is a promising drug target to improve the efficacy/safety window of antiplatelet agents. The native peptide GYPGQV, and the more-potent peptide AYPGKF, are PAR4-specific activators. However, these PAR4 agonist peptides (APs) elicit an agonist response, for example, platelet aggregation, at concentrations of 50 to 1000 µM in platelet-function assays, thereby limiting their utility to monitor the pharmacodynamic effects of PAR4 antagonists over a wide concentration range. Improved pharmacodynamic assays are needed for clinical development of PAR4 antagonists. We attempted to identify potent PAR4 APs to aid development of robust assays for optimization of PAR4 antagonists. Using an AYPG-based biased phage-display peptide library approach followed by chemical peptide optimization, A-Phe(4-F)-PGWLVKNG was identified. This peptide demonstrated an EC50 value of 3.4 µM in a platelet-aggregation assay, which is 16-fold more potent than AYPGKF. Using this new PAR4 AP, a platelet-rich plasma-aggregation assay using light-transmission aggregometry was developed and validated in a series of precision and reproducibility tests. PAR4 antagonist responses to PAR4 AP A-Phe(4-F)-PGWLVKNG (12.5 µM to 100 µM) were subsequently evaluated in this assay in vitro and ex vivo in a human study using BMS-986120, a PAR4 antagonist that entered clinical studies.

Reduced Apolipoprotein M and Adverse Outcomes Across the Spectrum of Human Heart Failure.

BACKGROUND: Apo (apolipoprotein) M mediates the physical interaction between high-density lipoprotein (HDL) particles and sphingosine-1-phosphate (S1P). Apo M exerts anti-inflammatory and cardioprotective effects in animal models. METHODS: In a subset of PHFS (Penn Heart Failure Study) participants (n=297), we measured apo M by Enzyme-Linked ImmunoSorbent Assay (ELISA). We also measured total S1P by liquid chromatography-mass spectrometry and isolated HDL particles to test the association between apo M and HDL-associated S1P. We confirmed the relationship between apo M and outcomes using modified aptamer-based apo M measurements among 2170 adults in the PHFS and 2 independent cohorts: the Washington University Heart Failure Registry (n=173) and a subset of TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial; n=218). Last, we examined the relationship between apo M and ≈5000 other proteins (SomaScan assay) to identify biological pathways associated with apo M in heart failure. RESULTS: In the PHFS, apo M was inversely associated with the risk of death (standardized hazard ratio, 0.56 [95% CI, 0.51-0.61]; P<0.0001) and the composite of death/ventricular assist device implantation/heart transplantation (standardized hazard ratio, 0.62 [95% CI, 0.58-0.67]; P<0.0001). This relationship was independent of HDL cholesterol or apo AI levels. Apo M remained associated with death (hazard ratio, 0.78 [95% CI, 0.69-0.88]; P<0.0001) and the composite of death/ventricular assist device/heart transplantation (hazard ratio, 0.85 [95% CI, 0.76-0.94]; P=0.001) in models that adjusted for multiple confounders. This association was present in both heart failure with reduced and preserved ejection fraction and was replicated in the Washington University cohort and a cohort with heart failure with preserved ejection fraction only (TOPCAT). The S1P and apo M content of isolated HDL particles strongly correlated (R=0.81, P<0.0001). The top canonical pathways associated with apo M were inflammation (negative association), the coagulation system (negative association), and liver X receptor/retinoid X receptor activation (positive association). The relationship with inflammation was validated with multiple inflammatory markers measured with independent assays. CONCLUSIONS: Reduced circulating apo M is independently associated with adverse outcomes across the spectrum of human heart failure. Further research is needed to assess whether the apo M/S1P axis is a suitable therapeutic target in heart failure.

Orally bioavailable amine-linked macrocyclic inhibitors of factor XIa.

The discovery of orally bioavailable FXIa inhibitors has been a challenge. Herein, we describe our efforts to address this challenge by optimization of our imidazole-based macrocyclic series. Our optimization strategy focused on modifications to the P2 prime, macrocyclic amide linker, and the imidazole scaffold. Replacing the amide of the macrocyclic linker with amide isosteres led to the discovery of substituted amine linkers which not only maintained FXIa binding affinity but also improved oral exposure in rats. Combining the optimized macrocyclic amine linker with a pyridine scaffold afforded compounds 23 and 24 that were orally bioavailable, single-digit nanomolar FXIa inhibitors with excellent selectivity against relevant blood coagulation enzymes.

Structure based design of macrocyclic factor XIa inhibitors: Discovery of cyclic P1 linker moieties with improved oral bioavailability.

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.

First-in-human study to assess the safety, pharmacokinetics and pharmacodynamics of BMS-962212, a direct, reversible, small molecule factor XIa inhibitor in non-Japanese and Japanese healthy subjects.

AIMS: The aims of the present study were to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of BMS-962212, a first-in-class factor XIa inhibitor, in Japanese and non-Japanese healthy subjects. METHODS: This was a randomized, placebo-controlled, double-blind, sequential, ascending-dose study of 2-h (part A) and 5-day (part B) intravenous (IV) infusions of BMS-962212. Part A used four doses (1.5, 4, 10 and 25 mg h-1 ) of BMS-962212 or placebo in a 6:2 ratio per dose. Part B used four doses (1, 3, 9 and 20 mg h-1 ) enrolling Japanese (n = 4 active, n = 1 placebo) and non-Japanese (n = 4 active, n = 1 placebo) subjects per dose. The PK, PD, safety and tolerability were assessed throughout the study. RESULTS: BMS-962212 was well tolerated; there were no signs of bleeding, and adverse events were mild. In parts A and B, BMS-962212 demonstrated dose proportionality. The mean half-life in parts A and B ranged from 2.04 to 4.94 h and 6.22 to 8.65 h, respectively. Exposure-dependent changes were observed in the PD parameters, activated partial thromboplastin time (aPTT) and factor XI clotting activity (FXI:C). The maximum mean aPTT and FXI:C change from baseline at 20 mg h-1 in part B was 92% and 90%, respectively. No difference was observed in weight-corrected steady-state concentrations, aPTT or FXI:C between Japanese and non-Japanese subjects (P > 0.05). CONCLUSION: BMS-962212 has tolerability, PK and PD properties suitable for investigational use as an acute antithrombotic agent in Japanese or non-Japanese subjects.

Pyridazine and pyridazinone derivatives as potent and selective factor XIa inhibitors.

Pyridazine and pyridazinone derivatives were designed and synthesized as coagulation factor XIa inhibitors. Potent and selective inhibitors with single digit nanomolar affinity for factor XIa were discovered. Selected inhibitors demonstrated moderate oral bioavailability.

Factor XIIa as a Novel Target for Thrombosis: Target Engagement Requirement and Efficacy in a Rabbit Model of Microembolic Signals.

Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin-4 (human FXIIa inhibition constant = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against Factor Xa (FXa) and plasmin. rHA-Mut-inf binds FXIIa, but not FXII zymogen, and competitively inhibits FXIIa protease activity. Its mode of action is hence akin to typical small-molecule inhibitors. Plasma shift and aPTT studies with rHA-Mut-inf demonstrated that calculated enzyme occupancy for FXIIa in achieving a putative aPTT doubling target in human, nonhuman primate, and rabbit is more than 99.0%. The effects of rHA-Mut-inf in carotid arterial thrombosis and microembolic signal (MES) in middle cerebral artery were assessed simultaneously in rabbits. Dose-dependent inhibition was observed for both arterial thrombosis and MES. The ED50 of thrombus formation was 0.17 mg/kg i.v. rHA-Mut-inf for the integrated blood flow and 0.16 mg/kg for thrombus weight; the ED50 for MES was 0.06 mg/kg. Ex vivo aPTT tracked with efficacy. In summary, our findings demonstrated that very high enzyme occupancy will be required for FXIIa active site inhibitors, highlighting the high potency and exquisite selectivity necessary for achieving efficacy in humans. Our MES studies suggest that targeting FXIIa may offer a promising strategy for stroke prevention associated with thromboembolic events.

Inhibition of Factor XIa Reduces the Frequency of Cerebral Microembolic Signals Derived from Carotid Arterial Thrombosis in Rabbits.

Factor XI (FXI) is an integral component of the intrinsic pathway of the coagulation cascade and plays a critical role in thrombus formation. Because its role in the pathogenesis of cerebral microembolic signals (MES) is unclear, this study used a potent and selective small molecule inhibitor of FXIa, compound 1, to assess the effect of FXI blockade in our recently established preclinical model of cerebral MES induced by FeCl3 injury of the carotid artery in male New Zealand White rabbits. Ascending doses of compound 1 were evaluated simultaneously for both carotid arterial thrombosis by a Doppler flowmeter and MES in the middle cerebral artery by a transcranial Doppler. Plasma drug exposure and pharmacodynamic responses to compound 1 treatment were also assessed. The effective dose for 50% inhibition (ED50) of thrombus formation was 0.003 mg/kg/h compound 1, i.v. for the integrated blood flow, 0.004 mg/kg/h for reduction in thrombus weight, and 0.106 mg/kg/h for prevention of MES. The highest dose, 3 mg/kg/h compound 1, achieved complete inhibition in both thrombus formation and MES. In addition, we assessed the potential bleeding liability of compound 1 (5 mg/kg/h, i.v., >1250-fold ED50 levels in arterial thrombosis) in rabbits using a cuticle bleeding model, and observed about 2-fold (not statistically significant) prolongation in bleeding time. Our study demonstrates that compound 1 produced a robust and dose-dependent inhibition of both arterial thrombosis and MES, suggesting that FXIa blockade may represent a novel therapeutic strategy for the reduction in MES in patients at risk for ischemic stroke.

Macrocyclic factor XIa inhibitors.

A series of macrocyclic factor XIa (FXIa) inhibitors was designed based on an analysis of the crystal structures of the acyclic phenylimidazole compounds. Further optimization using structure-based design led to inhibitors with pM affinity for FXIa, excellent selectivity against a panel of relevant serine proteases, and good potency in the activated partial thromboplastin time (aPTT) clotting assay.

Neutral macrocyclic factor VIIa inhibitors.

Factor VIIa (FVIIa) inhibitors have shown strong antithrombotic efficacy in preclinical thrombosis models with limited bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful due to the requirement of a basic P1 group to interact with Asp189 in the S1 binding pocket, limiting their membrane permeability. We have combined recently reported neutral P1 binding substituents with a highly optimized macrocyclic chemotype to produce FVIIa inhibitors with low nanomolar potency and enhanced permeability.

Macrocyclic inhibitors of Factor XIa: Discovery of alkyl-substituted macrocyclic amide linkers with improved potency.

Optimization of macrocyclic inhibitors of FXIa is described which focused on modifications to both the macrocyclic linker and the P1 group. Increases in potency were discovered through interactions with a key hydrophobic region near the S1 prime pocket by substitution of the macrocyclic linker with small alkyl groups. Both the position of substitution and the absolute stereochemistry of the alkyl groups on the macrocyclic linker which led to improved potency varied depending on the ring size of the macrocycle. Replacement of the chlorophenyltetrazole cinnamide P1 in these optimized macrocycles reduced the polar surface area and improved the oral bioavailability for the series, albeit at the cost of a decrease in potency.

Apixaban Inhibits Cerebral Microembolic Signals Derived from Carotid Arterial Thrombosis in Rabbits.

Cerebral microembolic signal (MES) is an independent predictor of stroke risk and prognosis. The objective of this study is to assess the effects of apixaban, as a representative of the novel oral anticoagulant class, on a rabbit model of cerebral MES. A clinical transcranial Doppler ultrasound instrument was used to assess MESs in the middle cerebral artery in a 30% FeCl3-induced carotid arterial thrombosis model in male New Zealand White rabbits. Ascending doses of apixaban were evaluated as monotherapy and in combination with aspirin on both arterial thrombosis and MES. Pharmacokinetic and pharmacodynamic responses were also evaluated. The effective dose for 50% inhibition (ED50) of thrombus formation for monotherapy was 0.04 mg/kg per hour apixaban, i.v. (0.03 µM plasma exposure) for the integrated blood flow, 0.13 mg/kg per hour apixaban (0.10 µM plasma exposure) for thrombus weight, and 0.03 mg/kg per hour apixaban (0.02 µM plasma exposure) for MES. Dual treatment with aspirin (5 mg/kg, PO) and apixaban (0.015 mg/kg per hour, i.v.) resulted in a significant reduction in cerebral MES (P < 0.05) compared with monotherapy with either agent. Pharmacokinetic analysis of apixaban and pharmacodynamic assays using activated partial thromboplastin time (aPTT) and prothrombin time (PT) for apixaban- and arachidonic acid-induced platelet aggregation for aspirin were used to confirm the exposure-response relationships. In summary, our study demonstrates that apixaban in a concentration-dependent manner inhibits both arterial thrombosis and MES, suggesting a potential association between factor Xa (FXa) blockade and the reduction in MES in patients at risk of ischemic stroke.

Synthesis and P1' SAR exploration of potent macrocyclic tissue factor-factor VIIa inhibitors.

Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1'-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1' group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1' substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

Novel phenylalanine derived diamides as Factor XIa inhibitors.

The synthesis, structural activity relationships (SAR), and selectivity profile of a potent series of phenylalanine diamide FXIa inhibitors will be discussed. Exploration of P1 prime and P2 prime groups led to the discovery of compounds with high FXIa affinity, good potency in our clotting assay (aPPT), and high selectivity against a panel of relevant serine proteases as exemplified by compound 21. Compound 21 demonstrated good in vivo efficacy (EC50=2.8µM) in the rabbit electrically induced carotid arterial thrombosis model (ECAT).

Orally bioavailable pyridine and pyrimidine-based Factor XIa inhibitors: Discovery of the methyl N-phenyl carbamate P2 prime group.

Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1, and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2 prime group which maintained FXIa activity, reduced the number of H-bond donors, and improved the physicochemical properties compared to the amino indazole P2 prime moiety. Compound (S)-17 was identified as a potent and selective FXIa inhibitor that was orally bioavailable. Replacement of the basic cyclohexyl methyl amine P1 in (S)-17 with the neutral p-chlorophenyltetrazole P1 resulted in the discovery of (S)-24 which showed a significant improvement in oral bioavailability compared to the previously reported imidazole (S)-23. Additional improvements in FXIa binding affinity, while maintaining oral bioavailability, was achieved by replacing the pyridine scaffold with either a regioisomeric pyridine or pyrimidine ring system.

Pyridine and pyridinone-based factor XIa inhibitors.

The structure-activity relationships (SAR) of six-membered ring replacements for the imidazole ring scaffold is described. This work led to the discovery of the potent and selective pyridine (S)-23 and pyridinone (±)-24 factor XIa inhibitors. SAR and X-ray crystal structure data highlight the key differences between imidazole and six-membered ring analogs.

Structure-based design of inhibitors of coagulation factor XIa with novel P1 moieties.

Compound 2 was previously identified as a potent inhibitor of factor XIa lacking oral bioavailability. A structure-based approach was used to design analogs of 2 with novel P1 moieties with good selectivity profiles and oral bioavailability. Further optimization of the P1 group led to the identification of a 4-chlorophenyltetrazole P1 analog, which when combined with further modifications to the linker and P2' group provided compound 32 with FXIa Ki=6.7 nM and modest oral exposure in dogs.

In vitro, antithrombotic and bleeding time studies of BMS-654457, a small-molecule, reversible and direct inhibitor of factor XIa.

BMS-654457 ((+) 3'-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-quinolin-2-yl)-4-carbamoyl-5'-(3-methyl-butyrylamino)-biphenyl-2-carboxylic acid) is a small-molecule factor XIa (FXIa) inhibitor. We evaluated the in vitro properties of BMS-654457 and its in vivo activities in rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). Kinetic studies conducted in vitro with a chromogenic substrate demonstrated that BMS-654457 is a reversible and competitive inhibitor for FXIa. BMS-654457 increased activated partial thromboplastin time (aPTT) without changing prothrombin time. It was equipotent in prolonging the plasma aPTT in human and rabbit, and less potent in rat and dog. It did not alter platelet aggregation to ADP, arachidonic acid and collagen. In vivo, BMS-654457 or vehicle was given IV prior to initiation of thrombosis or cuticle transection. Preservation of integrated carotid blood flow over 90 min (iCBF, % control) was used as a marker of antithrombotic efficacy. BMS-654457 at 0.37 mg/kg + 0.27 mg/kg/h produced almost 90 % preservation of iCBF compared to its vehicle (87 ± 10 and 16 ± 3 %, respectively, n = 6 per group) and increased BT by 1.2 ± 0.04-fold (P < 0.05). At a higher dose (1.1 mg/kg + 0.8 mg/kg/h), BMS-654457 increased BT by 1.33 ± 0.08-fold. This compares favorably to equivalent antithrombotic doses of reference anticoagulants (warfarin and dabigatran) and antiplatelet agents (clopidogrel and prasugrel) which produced four- to six-fold BT increases in the same model. In summary, BMS-654457 was effective in the prevention of arterial thrombosis in rabbits with limited effects on BT. This study supports inhibition of FXIa, with a small-molecule, reversible and direct inhibitor as a promising antithrombotic therapy with a wide therapeutic window.

Identification of 1-{2-[4-chloro-1'-(2,2-dimethylpropyl)-7-hydroxy-1,2-dihydrospiro[indole-3,4'-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and orally bioavailable P2Y(1) antagonist as an antiplatelet agent.

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.