Antithrombotic Effects of the Novel Small-Molecule Factor XIa Inhibitor Milvexian in a Rabbit Arteriovenous Shunt Model of Venous Thrombosis.

Background Factor XIa (FXIa) is an emerging therapeutic target, and FXIa inhibition is a promising mechanism to improve therapeutic index over current anticoagulants. Milvexian (BMS-986177/JNJ-70033093) is an oral small-molecule FXIa inhibitor. Objective Milvexian's antithrombotic efficacy was characterized in a rabbit arteriovenous (AV) shunt model of venous thrombosis and compared with the factor Xa inhibitor apixaban and the direct thrombin inhibitor dabigatran. Methods The AV shunt model of thrombosis was conducted in anesthetized rabbits. Vehicle or drugs were administered as intravenous bolus plus a continuous infusion. Thrombus weight was the primary efficacy endpoint. Ex vivo activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin time (TT) were measured as the pharmacodynamic responses. Results Milvexian dose dependently reduced thrombus weights by 34.3 ± 7.9, 51.6 ± 6.8 ( p < 0.01; n = 5), and 66.9 ± 4.8% ( p < 0.001; n = 6) versus vehicle at 0.25 + 0.17, 1.0 + 0.67, and 4.0 ± 2.68 mg/kg bolus + mg/kg/h infusion, respectively. Ex vivo clotting data supported a dose-dependent prolongation of aPTT (with 1.54-, 2.23-, and 3.12-fold increases from baseline upon the AV shunt start), but no changes in PT and TT. Dose-dependent inhibition in thrombus weight and clotting assays was also demonstrated for both apixaban and dabigatran as the references for the model validation. Conclusion Results demonstrate that milvexian is an effective anticoagulant for prevention of venous thrombosis in the rabbit model, which supports the utility of milvexian in venous thrombosis, as seen in the phase 2 clinical study.

Discovery of Potent and Orally Bioavailable Pyridine N-Oxide-Based Factor XIa Inhibitors through Exploiting Nonclassical Interactions.

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 (Ki = 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.

Lead Optimization to Advance Protease-Activated Receptor-1 Antagonists in Early Discovery.

Vorapaxar is an approved drug for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Subsequent to the discovery of Vorapaxar, medicinal chemistry efforts were continued to identify structurally differentiated leads. Toward this goal, extensive structure-activity relationship studies using a C-ring-truncated version of Vorapaxar culminated in the discovery of three leads, represented as 13, 14, and 23. Among these leads, compound 14 possessed favorable pharmacokinetic properties and an off-target profile, which supported additional profiling in an exploratory rat toxicology study.

Translational PK/PD and model-informed development of JNJ-67842125, a Fab reversal agent for JNJ-64179375, a long-acting thrombin inhibitor.

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.

Pharmacological Profile of JNJ-64179375: A Novel, Long-Acting Exosite-1 Thrombin Inhibitor.

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 α-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 EC2X values of 4.4, 12.4, 172.6, and 202.7 µg/ml, respectively. JNJ-9375 inhibited thrombin-induced platelet aggregation in human plasma with an IC50 value of 52.6 nM (7.8 µg/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 1.5× of TT. Bleeding was increased at 3 mg/kg in a rat tail transection bleeding model demonstrating a therapeutic index of 10× compared with 1× 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.

In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist.

Vorapaxar is a novel protease-activated receptor-1 (PAR1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. The present study provides a comprehensive in vitro pharmacological characterization of vorapaxar interaction with the PAR1 receptor on human platelets. Similar studies were performed with a metabolite of vorapaxar (M20). Vorapaxar and M20 were competitive PAR1 antagonists that demonstrated concentration-dependent, saturable, specific, and slowly reversible binding to the receptor present on intact human platelets. The affinities of vorapaxar and M20 for the PAR1 receptor were in the low nanomolar range, as determined by saturation-, kinetic- and competitive binding studies. The calculated Kd and Ki values for vorapaxar increased in the presence of plasma, indicating a decrease in the free fraction available for binding to the PAR1 receptor on human platelets. Vorapaxar was also evaluated in functional assays using thrombin or a PAR1 agonist peptide (SFLLRN). Vorapaxar and M20 completely blocked thrombin-stimulated PAR1/ß-arrestin association in recombinant cells and abolished thrombin-stimulated calcium influx in washed human platelets and vascular smooth muscle cells. Moreover, vorapaxar and M20 inhibited PAR1 agonist peptide-mediated platelet aggregation in human platelet rich plasma with a steep concentration response relationship. Vorapaxar exhibited high selectivity for inhibition of PAR1 over other platelet GPCRs. In conclusion, vorapaxar is a potent PAR1 antagonist exhibiting saturable, reversible, selective binding with slow off-rate kinetics and effectively inhibits thrombin's PAR1-mediated actions on human platelets.

Platelet transfusion reverses bleeding evoked by triple anti-platelet therapy including vorapaxar, a novel platelet thrombin receptor antagonist.

Vorapaxar is a novel protease-activated receptor-1 (PAR-1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Patients who received vorapaxar in addition to standard of care antiplatelet therapy had an increased incidence of major bleeding events compared with placebo. To assess whether platelet transfusion can restore hemostasis in primates on triple antiplatelet therapy, template bleeding times were assessed concurrently in the buccal mucosa, finger pad, and distolateral tail of anesthetized cynomolgus macaques to evaluate bleeding with vorapaxar as either monotherapy or in combination with aspirin or aspirin and clopidogrel. Aspirin (5mg/kg, IV) or vorapaxar (1mg/kg, PO) alone had no significant effect on bleeding times in the three vascular beds examined. A modest (<2-fold) increase in bleeding time was achieved in the three beds with the dual combination of aspirin and vorapaxar. Major increases in bleeding time were achieved in the three beds with the triple combination of aspirin (5mg/kg, IV), vorapaxar (1mg/kg, PO), and clopidogrel (1mg/kg, PO). Transfusion of fresh human platelet rich plasma, but not platelet poor plasma, reversed the increase in bleeding time in the triple therapy group. Transfusion of human platelets may be a viable approach in situations requiring a rapid reversal of platelet function in individuals treated with triple anti-platelet therapy that includes vorapaxar.

Iminopyrimidinones: a novel pharmacophore for the development of orally active renin inhibitors.

The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.

Himbacine-derived thrombin receptor antagonists: c7-aminomethyl and c9a-hydroxy analogues of vorapaxar.

We have synthesized several C7-aminomethyl analogues of vorapaxar that are potent PAR-1 antagonists. Many of these analogues showed excellent in vitro binding affinity and pharmacokinetics profile in rats. Compound 6a from this series showed excellent PAR-1 activity (K i = 5 nM). We have also synthesized a C9a-hydroxy analogue of vorapaxar, which showed very good PAR-1 affinity (K i = 19.5 nM) along with excellent rat pharmacokinetic profile and ex vivo efficacy in the cynomolgus monkey.

Himbacine-derived thrombin receptor antagonists: c7-spirocyclic analogues of vorapaxar.

We have synthesized several C7-spirocyclic analogues of vorapaxar and evaluated their in vitro activities against PAR-1 receptor. Some of these analogues showed activities and rat plasma levels comparable to vorapaxar. Compound 5c from this series showed excellent PAR-1 activity (K i = 5.1 nM). We also present a model of these spirocyclic compounds docked to the PAR-1 receptor based on the X-ray crystal structure of vorapaxar bound to PAR-1 receptor. This model explains some of the structure-activity relationships in this series.

Zucker Diabetic Fatty rats exhibit hypercoagulability and accelerated thrombus formation in the Arterio-Venous shunt model of thrombosis.

INTRODUCTION: Diabetes is a significant risk factor for thrombosis. The present study aimed at assessing coagulability, platelet reactivity, and thrombogenicity of the diabetic female Zucker Diabetic Fatty (ZDF) rat model and its relevance in studying antithrombotic mechanisms. MATERIALS AND METHODS: The basal coagulant state in ZDF rats was evaluated by clotting times, thromboelastography, and thrombin generation assay. A 14-day treatment with dapagliflozin in ZDF rats was pursued to investigate if glycemic control can improve coagulability. Thrombus formation in the Arterio-Venous (A-V) shunt model and the FeCl3-induced arterial thrombosis model was studied, with the antithrombotic effect of apixaban in the former model further investigated. RESULTS: ZDF rats exhibited significantly shortened clotting times, enhanced thrombin generation, and decreased fibrinolysis at baseline. Effective glycemic control achieved with dapagliflozin did not improve any of these parameters. ZDF rats displayed accelerated thrombus formation and were amenable to apixaban treatment in the A-V shunt model albeit with less sensitivity than normal rats. ZDF rats exhibited less platelet aggregation in response to ADP, collagen and PAR-4, and attenuated thrombotic response in the FeCl3 model. CONCLUSIONS: ZDF rats are at a chronic hypercoagulable and hypofibrinolytic state yet with compromised platelet reactivity. They display accelerated and attenuated thrombosis in the A-V shunt and FeCl3 model of thrombosis, respectively. Results shed new light on the pathophysiology of the ZDF rat model and illustrate its potential value in translational research on anticoagulant agents in diabetics. Caution needs to be exerted in utilizing this model in assessing antiplatelet mechanisms in diabetes-associated atherothrombosis.

A novel approach to assess the spontaneous gastrointestinal bleeding risk of antithrombotic agents using Apc(min/+) mice.

Assessment of the bleeding risk of antithrombotic agents is usually performed in healthy animals with some form of vascular injury to peripheral organs to induce bleeding. However, bleeding observed in patients with currently marketed antithrombotic drugs is typically spontaneous in nature such as intracranial haemorrhage (ICH) and gastrointestinal (GI) bleeding, which happens most frequently on top of preexisting pathologies such as GI ulcerations and polyps. Apc(min/+) mice are reported to develop multiple adenomas through the entire intestinal tract and display progressive anaemia.In this study, we evaluated the potential utility of Apc(min/+) mice as a model for assessing spontaneous GI bleeding with antithrombotic agents. Apc(min/+) mice exhibited progressive blood loss starting at the age of nine weeks. Despite the increase in bleeding, Apc(min/+) mice were in a hypercoagulable state and displayed an age-dependent increase in thrombin generation and circulating fibrinogen as well as a significant decrease in clotting times. We evaluated the effect of warfarin, dabigatran etexilate, apixaban and clopidogrel in this model by administering them in diet or in the drinking water to mice for 1-4 weeks. All of these marketed drugs significantly increased GI bleeding in Apc(min/+) mice, but not in wild-type mice. Although different exposure profiles of these antithrombotic agents make it challenging to compare the bleeding risk of compounds, our results indicate that the Apc(min/+) mouse may be a sensitive preclinical model for assessing the spontaneous GI bleeding risk of novel antithrombotic agents.

Differential profiles of thrombin inhibitors (heparin, hirudin, bivalirudin, and dabigatran) in the thrombin generation assay and thromboelastography in vitro.

Thrombin is a central enzyme in hemostasis and thrombosis, and a proven target for anticoagulant therapies. We compared four marketed and representative thrombin inhibitors, heparin, hirudin, bivalirudin, and dabigatran, in in-vitro spike-in assays that covered their therapeutic ranges. The assays employed were low tissue factor (1 pmol/l)-triggered thrombin generation assay (TGA) with plasma and 1:8000 Recombiplastin-triggered thromboelastography (TEG) with whole blood, with or without tissue plasminogen activator (tPA)-induced fibrinolysis. The three direct thrombin inhibitors (DTIs) prolonged TGA lag time and TEG clotting time (R) with a potency stack-ranking of hirudin>dabigatran approximately equal to bivalirudin. Heparin had the most steep concentration-response curve for both parameters. In TGA, 1-2 µmol/l dabigatran or hirudin resulted in complete inhibition on peak, slope, and endogenous thrombin potential, whereas bivalirudin had no effect on these parameters up to 10 µmol/l. All three DTIs, but not heparin, displayed a paradoxical increase in peak and slope in the low concentration range. In TEG, whereas all four agents reduced clot strength (maximal amplitude) in synergy with tPA, hirudin was the only DTI that reduced maximal amplitude appreciably without tPA. Dabigatran had the strongest potentiating effect on tPA-induced fibrinolytic activity (Ly30). With regard to the effects on coagulation and clot strength (lag time, R, and maximal amplitude) in the respective therapeutic range, dabigatran elicited the most modest changes. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. The contrasts between the agents are consistent with their known properties and are informative on efforts to define the optimal profiles of new anticoagulants.

Inhibition of protease-activated receptor 1 ameliorates intestinal radiation mucositis in a preclinical rat model.

PURPOSE: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. METHODS AND MATERIALS: Rats underwent fractionated X-irradiation (5 Gy×9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. RESULTS: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. CONCLUSION: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

Discovery of nor-seco himbacine analogs as thrombin receptor antagonists.

Discovery of a novel nor-seco himbacine analog as potent thrombin receptor (PAR-1) antagonist is described. Despite low plasma level, these new analogs showed excellent ex vivo efficacy in the monkey platelet aggregation assay. A potent hydroxy metabolite generated in vivo was identified as the agent responsible for the ex vivo efficacy. Following this discovery, the metabolite series was optimized to obtain analogs that showed very good ex vivo efficacy along with excellent pharmacokinetic profile in c. monkey.

SAR studies of C2 ethers of 2H-pyrano[2,3-d]pyrimidine-2,4,7(1H,3H)-triones as nicotinic acid receptor (NAR) agonist.

Based on in house screening lead compound 1 for the NAR project, SAR studies have been focused on the modification of the C2 ethers of the pyrimidinedione core structure. In this effort, an unpredictable SAR trend was overcome in the alkyl ether and arylalkyl ether series to identify compound 24 with improved in vitro activity compared to nicotinic acid. More consistent and predictable SAR was achieved in the propargyl ether series. Lead compound 41 was identified with good in vitro and in vivo activity in rat, and much improved rat PK profile.

Discovery of SCH 900271, a Potent Nicotinic Acid Receptor Agonist for the Treatment of Dyslipidemia.

Structure-guided optimization of a series of C-5 alkyl substituents led to the discovery of a potent nicotinic acid receptor agonist SCH 900271 (33) with an EC50 of 2 nM in the hu-GPR109a assay. Compound 33 demonstrated good oral bioavailability in all species. Compound 33 exhibited dose-dependent inhibition of plasma free fatty acid (FFA) with 50% FFA reduction at 1.0 mg/kg in fasted male beagle dogs. Compound 33 had no overt signs of flushing at doses up to 10 mg/kg with an improved therapeutic window to flushing as compared to nicotinic acid. Compound 33 was evaluated in human clinical trials.

Discovery of a potent nicotinic Acid receptor agonist for the treatment of dyslipidemia.

Nicotinic acid has been used clinically for decades to control serum lipoproteins. Nicotinic acid lowers very low-density lipoprotein (VLDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, and lipoprotein-a (LPa), and it is also effective in raising high-density lipoprotein (HDL)-cholesterol. However, nicotinic acid has several side effects in clinical use. The most notable is intense cutaneous vasodilation "flushing" on the upper body and face. We discovered a pyranopyrimidinedione series to be nicotinic acid receptor agonists. A potent nicotinic acid receptor agonist from this series {5-(3-cyclopropylpropyl)-2-(difluoromethyl)-3H-pyrano[2,3-d]pyrimidine-4,7-dione}with reduced flushing side effect in dogs was identified.

Discovery of a vorapaxar analog with increased aqueous solubility.

An analog of the thrombin receptor antagonist vorapaxar (SCH 530348) with increased aqueous solubility, compound 9c (SCH 602539), was discovered through incorporation of polar substituents on the pyridine ring of the himbacine-derived lead series. This analog retained the excellent potency, pharmacokinetic and safety properties of vorapaxar while increasing the aqueous solubility by 20-fold. Also presented are in vivo evaluations of this compound in a cynomolgus monkey platelet aggregation assay and in a Folts model of thrombosis in anesthetized monkeys.

SCH 602539, a protease-activated receptor-1 antagonist, inhibits thrombosis alone and in combination with cangrelor in a Folts model of arterial thrombosis in cynomolgus monkeys.

OBJECTIVE: To determine the antithrombotic effects of SCH 602539, an analog of the selective protease-activated receptor (PAR)-1 antagonist vorapaxar (formerly SCH 530348) currently in advanced clinical development, and the P2Y(12) ADP receptor antagonist cangrelor, alone and in combination. METHODS AND RESULTS: Multiple platelet activation pathways contribute to thrombosis. The effects of SCH 602539 and cangrelor alone and in combination on cyclic flow reductions were evaluated in a Folts model of thrombosis in cynomolgus monkeys. The effects of these treatments on ex vivo platelet aggregation and coagulation parameters were also monitored. Dose-dependent inhibition of cyclic flow reductions was observed after treatment with SCH 602539 alone and cangrelor alone (P<0.05 versus vehicle for the 2 highest concentrations of each agent). The combination of SCH 602539 and cangrelor was associated with synergistic antithrombotic effects (P<0.05 versus vehicle for all combinations tested). The 2 highest doses of SCH 602539 inhibited platelet aggregation in response to PAR-1-selective high-affinity thrombin receptor agonist peptide by greater than 80% but did not affect platelet aggregation induced by other agonists; also, they did not affect any coagulation parameters. CONCLUSIONS: The combined inhibition of the PAR-1 and the P2Y(12) ADP platelet activation pathways had synergistic antithrombotic and antiplatelet effects. The addition of a PAR-1 antagonist to a P2Y(12) ADP receptor antagonist may provide incremental clinical benefits in patients with atherothrombotic disease, both in short- and long-term settings. These hypotheses need to be tested clinically.