First-in-human study of milvexian, an oral, direct, small molecule factor XIa inhibitor.

Milvexian (BMS-986177/JNJ-70033093) is a small molecule, active-site inhibitor of factor XIa (FXIa) being developed to prevent and treat thrombotic events. The safety, tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of milvexian were assessed in a two-part, double-blind, placebo-controlled, sequential single ascending dose (SAD) and multiple ascending dose (MAD) study in healthy adults. Participants in SAD panels (6 panels of 8 participants; n = 48) were randomized (3:1) to receive milvexian (4, 20, 60, 200, 300, or 500 mg) or placebo. The 200- and 500-mg panels investigated the pharmacokinetic impact of a high-fat meal. Participants in MAD panels (7 panels of 8 participants; n = 56) were randomized (3:1) to receive milvexian (once- or twice-daily) or placebo for 14 days. All milvexian dosing regimens were safe and well-tolerated, with only mild treatment-emergent adverse events and no clinically significant bleeding events. In SAD panels, maximum milvexian plasma concentration occurred 3 h postdose in all fasted panels. The terminal half-life (T1/2 ) ranged from 8.3 to 13.8 h. In fasted panels from 20 to 200 mg, absorption was dose-proportional; results at higher doses (300 and 500 mg) were consistent with saturable absorption. Food increased milvexian bioavailability in a dose-dependent fashion. In MAD panels, steady-state milvexian plasma concentration was reached within 3 and 6 dosing days with once- and twice-daily dosing, respectively. Renal excretion was less than 20% in all panels. Prolongation of activated partial thromboplastin time was observed and was directly related to drug exposure. These results suggest that the safety, tolerability, PK, and PD properties of milvexian are suitable for further clinical development.

Discovery of Milvexian, a High-Affinity, Orally Bioavailable Inhibitor of Factor XIa in Clinical Studies for Antithrombotic Therapy.

Factor XIa (FXIa) is an enzyme in the coagulation cascade thought to amplify thrombin generation but has a limited role in hemostasis. From preclinical models and human genetics, an inhibitor of FXIa has the potential to be an antithrombotic agent with superior efficacy and safety. Reversible and irreversible inhibitors of FXIa have demonstrated excellent antithrombotic efficacy without increased bleeding time in animal models (Weitz, J. I., Chan, N. C. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (1), 7-12). Herein, we report the discovery of a novel series of macrocyclic FXIa inhibitors containing a pyrazole P2' moiety. Optimization of the series for (pharmacokinetic) PK properties, free fraction, and solubility resulted in the identification of milvexian (BMS-986177/JNJ-70033093, 17, FXIa Ki = 0.11 nM) as a clinical candidate for the prevention and treatment of thromboembolic disorders, suitable for oral administration.

Milvexian, an orally bioavailable, small-molecule, reversible, direct inhibitor of factor XIa: In vitro studies and in vivo evaluation in experimental thrombosis in rabbits.

BACKGROUND: Milvexian (BMS-986177/JNJ-70033093) is an orally bioavailable factor XIa (FXIa) inhibitor currently in phase 2 clinical trials. OBJECTIVES: To evaluate in vitro properties and in vivo characteristics of milvexian. METHODS: In vitro properties of milvexian were evaluated with coagulation and enzyme assays, and in vivo profiles were characterized with rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). RESULTS: Milvexian is an active-site, reversible inhibitor of human and rabbit FXIa (Ki 0.11 and 0.38 nM, respectively). Milvexian increased activated partial thromboplastin time (APTT) without changing prothrombin time and potently prolonged plasma APTT in humans and rabbits. Milvexian did not alter platelet aggregation to ADP, arachidonic acid, or collagen. Milvexian was evaluated for in vivo prevention and treatment of thrombosis. For prevention, milvexian 0.063 + 0.04, 0.25 + 0.17, and 1 + 0.67 mg/kg+mg/kg/h preserved 32 ± 6*, 54 ± 10*, and 76 ± 5%* of carotid blood flow (CBF) and reduced thrombus weight by 15 ± 10*, 45 ± 2*, and 70 ± 4%*, respectively (*p < .05; n = 6/dose). For treatment, thrombosis was initiated for 15 min and CBF decreased to 40% of control. Seventy-five minutes after milvexian administration, CBF averaged 1 ± 0.3, 39 ± 10, and 66 ± 2%* in groups treated with vehicle and milvexian 0.25 + 0.17 and 1 + 0.67 mg/kg+mg/kg/h, respectively (*p < .05 vs. vehicle; n = 6/group). The combination of milvexian 1 + 0.67 mg/kg+mg/kg/h and aspirin 4 mg/kg/h intravenous did not increase BT versus aspirin monotherapy. CONCLUSIONS: Milvexian is an effective antithrombotic agent with limited impact on hemostasis, even when combined with aspirin in rabbits. This study supports inhibition of FXIa with milvexian as a promising antithrombotic therapy with a wide therapeutic window.

Plasma biomarkers associated with adverse outcomes in patients with calcific aortic stenosis.

AIMS: Enhanced risk stratification of patients with aortic stenosis (AS) is necessary to identify patients at high risk for adverse outcomes, and may allow for better management of patient subgroups at high risk of myocardial damage. The objective of this study was to identify plasma biomarkers and multimarker profiles associated with adverse outcomes in AS. METHODS AND RESULTS: We studied 708 patients with calcific AS and measured 49 biomarkers using a Luminex platform. We studied the correlation between biomarkers and the risk of (i) death and (ii) death or heart failure-related hospital admission (DHFA). We also utilized machine-learning methods (a tree-based pipeline optimizer platform) to develop multimarker models associated with the risk of death and DHFA. In this cohort with a median follow-up of 2.8 years, multiple biomarkers were significantly predictive of death in analyses adjusted for clinical confounders, including tumour necrosis factor (TNF)-α [hazard ratio (HR) 1.28, P < 0.0001], TNF receptor 1 (TNFRSF1A; HR 1.38, P < 0.0001), fibroblast growth factor (FGF)-23 (HR 1.22, P < 0.0001), N-terminal pro B-type natriuretic peptide (NT-proBNP) (HR 1.58, P < 0.0001), matrix metalloproteinase-7 (HR 1.24, P = 0.0002), syndecan-1 (HR 1.27, P = 0.0002), suppression of tumorigenicity-2 (ST2) (IL1RL1; HR 1.22, P = 0.0002), interleukin (IL)-8 (CXCL8; HR 1.22, P = 0.0005), pentraxin (PTX)-3 (HR 1.17, P = 0.001), neutrophil gelatinase-associated lipocalin (LCN2; HR 1.18, P < 0.0001), osteoprotegerin (OPG) (TNFRSF11B; HR 1.26, P = 0.0002), and endostatin (COL18A1; HR 1.28, P = 0.0012). Several biomarkers were also significantly predictive of DHFA in adjusted analyses including FGF-23 (HR 1.36, P < 0.0001), TNF-α (HR 1.26, P < 0.0001), TNFR1 (HR 1.34, P < 0.0001), angiopoietin-2 (HR 1.26, P < 0.0001), syndecan-1 (HR 1.23, P = 0.0006), ST2 (HR 1.27, P < 0.0001), IL-8 (HR 1.18, P = 0.0009), PTX-3 (HR 1.18, P = 0.0002), OPG (HR 1.20, P = 0.0013), and NT-proBNP (HR 1.63, P < 0.0001). Machine-learning multimarker models were strongly associated with adverse outcomes (mean 1-year probability of death of 0%, 2%, and 60%; mean 1-year probability of DHFA of 0%, 4%, 97%; P < 0.0001). In these models, IL-6 (a biomarker of inflammation) and FGF-23 (a biomarker of calcification) emerged as the biomarkers of highest importance. CONCLUSIONS: Plasma biomarkers are strongly associated with the risk of adverse outcomes in patients with AS. Biomarkers of inflammation and calcification were most strongly related to prognosis.

Preclinical metabolism and disposition of an orally bioavailable macrocyclic FXIa inhibitor.

FXIa-6f is a high affinity, orally bioavailable macrocyclic FXIa inhibitor with antithrombotic activity in preclinical species.The objectives of this study were to characterize the in vitro metabolism, determine circulating metabolites in pre-clinical species, and examine the disposition of the compound in a bile duct-cannulated rat study (BDC) study to inform clinical development of the compound and the medicinal chemistry approach to identify molecules with improved properties.Across species, metabolic pathways included several oxidative metabolites, including hydroxylated metabolites on the macrocycle or P1 region, descarbamoylation of the methyl carbamate side chain, and a glutathione conjugate on the 2,6-difluoro-3-chlorophenyl ring.In BDC rat, the absorbed dose of [3H]FXIa-6f was cleared mainly by metabolism, with excretion of drug-related material in the bile, mostly as metabolites.In all preclinical species, the parent drug was the primary drug-related component in circulation, but the species differences in the metabolic pathways observed in vitro were reflected in the plasma, where M6, a descarbamoylated metabolite, was more prominent in rat plasma, and M9, a hydroxylated metabolite, was more prominent in monkey plasma. Based on the available data, the human metabolism appears to be most similar to monkey.

Antithrombotic Effects of Combined PAR (Protease-Activated Receptor)-4 Antagonism and Factor Xa Inhibition.

OBJECTIVE: PAR (protease-activated receptor)-4 antagonism has antiplatelet effects under conditions of high shear stress. We aimed to establish whether PAR4 antagonism had additive antithrombotic activity in the presence of factor Xa inhibition in an ex vivo model of acute arterial injury. Approach and Results: Fifteen healthy volunteers (29±6 years, 7 women) completed a phase zero double-blind randomized controlled crossover trial. Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured following blood perfusion of low shear and high shear stress chambers. Upstream of the chambers, extracorporeal blood was admixed with (1) vehicle, (2) low-dose apixaban (20 ng/mL), (3) high-dose apixaban (80 ng/mL), (4) BMS-986141 (400 ng/mL), (5) BMS-968141 and low-dose apixaban, or (6) BMS-968141 and high-dose apixaban in 6 sequential studies performed in random order. Compared with vehicle, BMS-986141 demonstrated selective inhibition of PAR4-AP (agonist peptide)-stimulated platelet aggregation, platelet-monocyte aggregates, and P-selectin expression (P≤0.01 for all). Total thrombus area was reduced under both low shear and high shear stress conditions for all drug infusions (P<0.0001 for all versus vehicle). BMS-968141 reduced total (≤44.4%) and platelet-rich (≤39.3%) thrombus area, whereas apixaban reduced total (≤42.9%) and fibrin-rich (≤31.6%) thrombus area. Combination of BMS-986141 with apixaban caused a further modest reduction in total thrombus area (9.6%-12.4%), especially under conditions of high shear stress (P≤0.027). CONCLUSIONS: In the presence of factor Xa inhibition, PAR4 antagonism with BMS-986141 further reduces thrombus formation, especially under conditions of high shear stress. This suggests the potential for additive efficacy of combination PAR4 antagonism and factor Xa inhibition in the prevention of atherothrombotic events.

Discovery of a High Affinity, Orally Bioavailable Macrocyclic FXIa Inhibitor with Antithrombotic Activity in Preclinical Species.

Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we report application of a previously disclosed cyclic carbamate P1 linker which provided improved oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency and good oral bioavailability but high in vivo clearance. Further structure-activity relationship (SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with selectivity against most of the relevant serine proteases. Compound 6f also demonstrated excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy model at doses which preserved hemostasis.

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.

Potent, Orally Bioavailable, and Efficacious Macrocyclic Inhibitors of Factor XIa. Discovery of Pyridine-Based Macrocycles Possessing Phenylazole Carboxamide P1 Groups.

Factor XIa (FXIa) inhibitors are promising novel anticoagulants, which show excellent efficacy in preclinical thrombosis models with minimal effects on hemostasis. The discovery of potent and selective FXIa inhibitors which are also orally bioavailable has been a challenge. Here, we describe optimization of the imidazole-based macrocyclic series and our initial progress toward meeting this challenge. A two-pronged strategy, which focused on replacement of the imidazole scaffold and the design of new P1 groups, led to the discovery of potent, orally bioavailable pyridine-based macrocyclic FXIa inhibitors. Moreover, pyridine-based macrocycle 19, possessing the phenylimidazole carboxamide P1, exhibited excellent selectivity against relevant blood coagulation enzymes and displayed antithrombotic efficacy in a rabbit thrombosis model.

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.

Factor XIa Inhibitors as New Anticoagulants.

With the introduction of thrombin and factor Xa inhibitors to the oral anticoagulant market, significant improvements in both efficacy and safety have been achieved. Early clinical and preclinical data suggest that inhibitors of factor XIa can provide a still safer alternative, with expanded efficacy for arterial indications. This Perspective provides an overview of target rationale and details of the discovery and development of inhibitors of factor XIa as next generation antithrombotic agents.

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.

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.

Discovery of a Parenteral Small Molecule Coagulation Factor XIa Inhibitor Clinical Candidate (BMS-962212).

Factor XIa (FXIa) is a blood coagulation enzyme that is involved in the amplification of thrombin generation. Mounting evidence suggests that direct inhibition of FXIa can block pathologic thrombus formation while preserving normal hemostasis. Preclinical studies using a variety of approaches to reduce FXIa activity, including direct inhibitors of FXIa, have demonstrated good antithrombotic efficacy without increasing bleeding. On the basis of this potential, we targeted our efforts at identifying potent inhibitors of FXIa with a focus on discovering an acute antithrombotic agent for use in a hospital setting. Herein we describe the discovery of a potent FXIa clinical candidate, 55 (FXIa Ki = 0.7 nM), with excellent preclinical efficacy in thrombosis models and aqueous solubility suitable for intravenous administration. BMS-962212 is a reversible, direct, and highly selective small molecule inhibitor of FXIa.

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.

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.

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.

Design and Synthesis of Novel Meta-Linked Phenylglycine Macrocyclic FVIIa Inhibitors.

Two novel series of meta-linked phenylglycine-based macrocyclic FVIIa inhibitors have been designed to improve the rodent metabolic stability and PK observed with the precursor para-linked phenylglycine macrocycles. Through iterative structure-based design and optimization, the TF/FVIIa Ki was improved to subnanomolar levels with good clotting activity, metabolic stability, and permeability.

Structure-Based Design of Macrocyclic Factor XIa Inhibitors: Discovery of the Macrocyclic Amide Linker.

A novel series of macrocyclic FXIa inhibitors was designed based on our lead acyclic phenyl imidazole chemotype. Our initial macrocycles, which were double-digit nanomolar FXIa inhibitors, were further optimized with assistance from utilization of structure-based drug design and ligand bound X-ray crystal structures. This effort resulted in the discovery of a macrocyclic amide linker which was found to form a key hydrogen bond with the carbonyl of Leu41 in the FXIa active site, resulting in potent FXIa inhibitors. The macrocyclic FXIa series, exemplified by compound 16, had a FXIa Ki = 0.16 nM with potent anticoagulant activity in an in vitro clotting assay (aPTT EC1.5x = 0.27 µM) and excellent selectivity against the relevant blood coagulation enzymes.

Discovery of Phenylglycine Lactams as Potent Neutral Factor VIIa Inhibitors.

Inhibitors of Factor VIIa (FVIIa), a serine protease in the clotting cascade, have shown strong antithrombotic efficacy in preclinical thrombosis models with minimal bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful because known chemotypes have required a highly basic group in the S1 binding pocket for high affinity. A recently reported fragment screening effort resulted in the discovery of a neutral heterocycle, 7-chloro-3,4-dihydroisoquinolin-1(2H)-one, that binds in the S1 pocket of FVIIa and can be incorporated into a phenylglycine FVIIa inhibitor. Optimization of this P1 binding group led to the first series of neutral, permeable FVIIa inhibitors with low nanomolar potency.