ABSTRACT
Apixaban is a factor Xa (FXa) inhibitor and standard-of-care anticoagulant with FXa Ki and plasma protein binding (free fraction) averages 0.08 nM and 0.13 in humans and 0.16 nM and 0.37 in rabbits, respectively. Apixaban at the approved dose of 5 mg BID achieved maximum and minimum plasma concentration of 373 nM (95% CI: 198 - 699 nM) and 224 nM (95% CI 89-501 nM), respectively, in patients with nonvalvular atrial fibrillation (AF). We calibrated the rabbit model of electrolytic-mediated arterial thrombosis (ECAT) against apixaban and correlated the potencies derived from the rabbit ECAT to in vivo efficacious exposure levels in AF patients. Vehicle and apixaban at multiple doses were infused IV in ECAT rabbits and their effects on thrombus weight were measured. Apixaban exhibited dose-related efficacy in preventing thrombosis in ECAT rabbits with EC20 , EC50 , EC60 , EC70 and EC80 of 18, 101, 169, 296, and 585 nM, respectively. After correcting for the human-to-rabbit potency based on FXa Ki and plasma protein binding, we estimated a rabbit-equally-effective plasma concentration of 157 and 259 nM to the trough and peak plasma concentration in AF patients treated with 5 mg BID of apixaban. These rabbit-equally-effective plasma concentrations matched well with the rabbit ECAT EC60 and EC70 . This study supports the potential of the rabbit ECAT to predict in vivo therapeutic drug exposure of FXa inhibitors. Achieving human-equally-effective plasma concentrations to the rabbit ECAT EC60 and EC70 may produce clinical efficacy in patient populations like AF.
Subject(s)
Anticoagulants , Thrombosis , Animals , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Calibration , Factor Xa Inhibitors/pharmacology , Factor Xa Inhibitors/therapeutic use , Humans , Pyrazoles , Pyridones , Rabbits , Thrombosis/drug therapy , Thrombosis/prevention & controlABSTRACT
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.
Subject(s)
Carotid Artery Thrombosis , Thrombosis , Animals , Carotid Artery Thrombosis/drug therapy , Factor XIa , Fibrinolytic Agents/therapeutic use , Partial Thromboplastin Time , Rabbits , Thrombosis/drug therapyABSTRACT
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.
Subject(s)
Carotid Artery Thrombosis , Factor XIa , Fibrinolytic Agents , Pyrimidines , Triazoles , Animals , Mice , Rabbits , Administration, Oral , Carotid Artery Thrombosis/drug therapy , Factor XIa/antagonists & inhibitors , Fibrinolytic Agents/administration & dosage , Fibrinolytic Agents/chemical synthesis , Fibrinolytic Agents/pharmacokinetics , Fibrinolytic Agents/therapeutic use , Macaca fascicularis , Molecular Structure , Pyrazoles/administration & dosage , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Pyrazoles/therapeutic use , Pyrimidines/administration & dosage , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Rats, Sprague-Dawley , Structure-Activity Relationship , Triazoles/administration & dosage , Triazoles/chemical synthesis , Triazoles/pharmacokinetics , Triazoles/therapeutic useABSTRACT
BACKGROUND: New heart failure therapies that safely augment cardiac contractility and output are needed. Previous apelin peptide studies have highlighted the potential for APJ (apelin receptor) agonism to enhance cardiac function in heart failure. However, apelin's short half-life limits its therapeutic utility. Here, we describe the preclinical characterization of a novel, orally bioavailable APJ agonist, BMS-986224. METHODS: BMS-986224 pharmacology was compared with (Pyr1) apelin-13 using radio ligand binding and signaling pathway assays downstream of APJ (cAMP, phosphorylated ERK [extracellular signal-regulated kinase], bioluminescence resonance energy transfer-based G-protein assays, ß-arrestin recruitment, and receptor internalization). Acute effects on cardiac function were studied in anesthetized instrumented rats. Chronic effects of BMS-986224 were assessed echocardiographically in the RHR (renal hypertensive rat) model of cardiac hypertrophy and decreased cardiac output. RESULTS: BMS-986224 was a potent (Kd=0.3 nmol/L) and selective APJ agonist, exhibiting similar receptor binding and signaling profile to (Pyr1) apelin-13. G-protein signaling assays in human embryonic kidney 293 cells and human cardiomyocytes confirmed this and demonstrated a lack of signaling bias relative to (Pyr1) apelin-13. In anesthetized instrumented rats, short-term BMS-986224 infusion increased cardiac output (10%-15%) without affecting heart rate, which was similar to (Pyr1) apelin-13 but differentiated from dobutamine. Subcutaneous and oral BMS-986224 administration in the RHR model increased stroke volume and cardiac output to levels seen in healthy animals but without preventing cardiac hypertrophy and fibrosis, effects differentiated from enalapril. CONCLUSIONS: We identify a novel, potent, and orally bioavailable nonpeptidic APJ agonist that closely recapitulates the signaling properties of (Pyr1) apelin-13. We show that oral APJ agonist administration induces a sustained increase in cardiac output in the cardiac disease setting and exhibits a differentiated profile from the renin-angiotensin system inhibitor enalapril, supporting further clinical evaluation of BMS-986224 in heart failure.
Subject(s)
Apelin Receptors/agonists , Cardiac Output/drug effects , Heart Failure/physiopathology , Intercellular Signaling Peptides and Proteins/pharmacology , Stroke Volume/drug effects , Animals , Bioluminescence Resonance Energy Transfer Techniques , CHO Cells , Cricetulus , Dogs , Extracellular Signal-Regulated MAP Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , HEK293 Cells , Haplorhini , Humans , In Vitro Techniques , MAP Kinase Signaling System/drug effects , Phosphorylation , Radioligand Assay , Rats , Tritium , Ventricular Pressure/drug effects , beta-Arrestins/drug effects , beta-Arrestins/metabolismABSTRACT
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.
Subject(s)
Factor XIa/antagonists & inhibitors , Fibrinolytic Agents/administration & dosage , Fibrinolytic Agents/pharmacology , Administration, Oral , Animals , Biological Availability , Crystallography, X-Ray , Dogs , Drug Evaluation, Preclinical , Factor XIa/chemistry , Factor XIa/metabolism , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacokinetics , Humans , Hydrophobic and Hydrophilic Interactions , Macrocyclic Compounds/administration & dosage , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacokinetics , Macrocyclic Compounds/pharmacology , Models, Molecular , Rabbits , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Factor XIa/antagonists & inhibitors , Fibrinolytic Agents/chemical synthesis , Fibrinolytic Agents/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Animals , Biological Availability , Blood Coagulation/drug effects , Crystallography, X-Ray , Drug Design , Drug Discovery , Fibrinolytic Agents/pharmacokinetics , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/pharmacology , Models, Molecular , Partial Thromboplastin Time , Rabbits , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Thrombosis/drug therapyABSTRACT
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.
Subject(s)
Anticoagulants/chemistry , Anticoagulants/therapeutic use , Factor XIa/antagonists & inhibitors , Isoquinolines/chemistry , Isoquinolines/therapeutic use , Thrombosis/drug therapy , para-Aminobenzoates/chemistry , para-Aminobenzoates/therapeutic use , Animals , Anticoagulants/pharmacokinetics , Anticoagulants/pharmacology , Blood Coagulation/drug effects , Crystallography, X-Ray , Dogs , Drug Discovery , Factor XIa/chemistry , Factor XIa/metabolism , Humans , Isoquinolines/pharmacokinetics , Isoquinolines/pharmacology , Male , Molecular Docking Simulation , Rabbits , Rats , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic use , Thrombosis/blood , para-Aminobenzoates/pharmacokinetics , para-Aminobenzoates/pharmacologyABSTRACT
Adenosine diphosphate directly induces platelet aggregation via the G-protein coupled P2Y1 and P2Y12 receptors. P2Y12, but not P2Y1, receptor antagonists are available in the clinic. The relevance of the P2Y1 receptor as an antiplatelet target has been studied in rodents, but not in higher species. We therefore examined effects of the pharmacological blockade of the P2Y1 receptor with its selective antagonist MRS2500 in monkey models of electrolytic-mediated arterial thrombosis (ECAT) and kidney bleeding time (KBT). Abciximab, a GPIIb-IIIa antagonist, and cangrelor, a P2Y12 antagonist, were utilized to validate these monkey models. Compounds were given IV at 15-60 min before thrombosis initiation in anesthetized monkeys. Scanning electron microscopy showed the luminal surface of thrombotic artery covered with platelet aggregates and fibrin network. Administration of abciximab at 0.25 and 0.7 mg/kg IV significantly reduced thrombus weight by 71 ± 1 and 100 ± 0 %, and increased KBT by 10.0 ± 0.1- and 10.1 ± 0-fold, respectively (n = 3/dose). Likewise, cangrelor at 0.6 and 2 mg/kg/h IV significantly reduced thrombus weight significantly by 72 ± 9 % and 100 ± 0 % and increased KBT by 2.1 ± 0.1- and 9.8 ± 0.2-fold, respectively (n = 3/dose). MRS2500 [mg/kg + mg/kg/h IV] at 0.09 + 0.14 and 0.45 + 0.68 significantly reduced thrombus weight by 57 ± 1 % and 88 ± 1 % and increased KBT by 2.1 ± 0.3- and 4.9 ± 0.6-fold, respectively (n = 4/dose). In summary, MRS2500 prevented occlusive arterial thrombosis at a dose that moderately prolonged KBT, indicating a role of P2Y1 receptors in arterial thrombosis and hemostasis in monkeys. Thus P2Y1 receptor antagonism provides a suitable target for drug discovery.
Subject(s)
Carotid Arteries , Deoxyadenine Nucleotides/pharmacology , Purinergic P2Y Receptor Agonists/pharmacology , Thrombosis/prevention & control , Animals , Drug Evaluation, Preclinical , Macaca fascicularisABSTRACT
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).
Subject(s)
Anilides/pharmacology , Factor XIa/antagonists & inhibitors , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Anilides/chemical synthesis , Animals , Crystallography, X-Ray , Dogs , Phenylalanine/chemical synthesis , Rabbits , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Factor XIa/antagonists & inhibitors , Fibrinolytic Agents/pharmacology , Thrombosis/drug therapy , Animals , Bleeding Time , Dogs , Fibrinolytic Agents/chemistry , Humans , Partial Thromboplastin Time , Rabbits , Rats , Species Specificity , Thrombosis/bloodABSTRACT
Structure-activity relationship optimization of phenylalanine P1' and P2' regions with a phenylimidazole core resulted in a series of potent FXIa inhibitors. Introducing 4-hydroxyquinolin-2-one as the P2' group enhanced FXIa affinity and metabolic stability. Incorporation of an N-methyl piperazine amide group to replace the phenylalanine improved both FXIa potency and aqueous solubility. Combination of the optimization led to the discovery of FXIa inhibitor 13 with a FXIa K i of 0.04 nM and an aPTT EC2x of 1.0 µM. Dose-dependent efficacy (EC50 of 0.53 µM) was achieved in the rabbit ECAT model with minimal bleeding time prolongation.
ABSTRACT
Adenosine diphosphate (ADP)-mediated platelet aggregation is signaled through two distinct G protein-coupled receptors (GPCR) on the platelet surface: P2Y12 and P2Y1. Blocking P2Y12 receptor is a clinically well-validated strategy for antithrombotic therapy. P2Y1 antagonists have been shown to have the potential to provide equivalent antithrombotic efficacy as P2Y12 inhibitors with reduced bleeding in preclinical animal models. We have previously reported the discovery of a potent and orally bioavailable P2Y1 antagonist, 1. This paper describes further optimization of 1 by introducing 4-aryl groups at the hydroxylindoline in two series. In the neutral series, 10q was identified with excellent potency and desirable pharmacokinetic (PK) profile. It also demonstrated similar antithrombotic efficacy with less bleeding compared with the known P2Y12 antagonist prasugrel in rabbit efficacy/bleeding models. In the basic series, 20c (BMS-884775) was discovered with an improved PK and liability profile over 1. These results support P2Y1 antagonism as a promising new antiplatelet target.
Subject(s)
Drug Discovery , Indoles/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Purinergic P2Y Receptor Antagonists/pharmacology , Receptors, Purinergic P2Y1/metabolism , Animals , Blood Coagulation/drug effects , Dose-Response Relationship, Drug , Humans , Indoles/chemistry , Mice , Microsomes, Liver/chemistry , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Structure , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/chemical synthesis , Platelet Aggregation Inhibitors/chemistry , Purinergic P2Y Receptor Antagonists/chemical synthesis , Purinergic P2Y Receptor Antagonists/chemistry , Rabbits , Rats , Structure-Activity Relationship , Thrombosis/drug therapyABSTRACT
Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.
Subject(s)
Fibrinolytic Agents/chemistry , Phenylurea Compounds/chemistry , Purinergic P2Y Receptor Antagonists/chemistry , Pyridines/chemistry , Receptors, Purinergic P2Y1/chemistry , Urea/chemistry , Animals , Caco-2 Cells , Disease Models, Animal , Drug Evaluation, Preclinical , Fibrinolytic Agents/chemical synthesis , Fibrinolytic Agents/pharmacokinetics , Half-Life , Humans , Microsomes, Liver/metabolism , Partial Thromboplastin Time , Phenylurea Compounds/pharmacokinetics , Phenylurea Compounds/therapeutic use , Platelet Aggregation/drug effects , Purinergic P2Y Receptor Antagonists/pharmacokinetics , Purinergic P2Y Receptor Antagonists/therapeutic use , Pyridines/pharmacokinetics , Pyridines/therapeutic use , Rabbits , Rats , Receptors, Purinergic P2Y1/metabolism , Solubility , Structure-Activity Relationship , Thrombosis/drug therapy , Urea/pharmacokinetics , Urea/therapeutic use , Water/chemistryABSTRACT
BMS-262084 is a 4-carboxy-2-azetidinone-containing irreversible inhibitor of FXIa, which is selective over other coagulation proteases. We evaluated the in vitro and in vivo properties of BMS-262084 in rabbits. Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrolytic-mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. BMS-262084 was infused IV from 1 h before thrombus induction or cuticle transection to the end of the experiment. In vitro, BMS-262084 prolonged activated partial thromboplastin time (aPTT) with EC(2x) (concentration required to double aPTT) of 10.6 µM in rabbit plasma, and did not prolong prothrombin time (PT), thrombin time (TT) and HepTest. In vivo, BMS-262084 produced dose-dependent antithrombotic effects in rabbits with antithrombotic ED(50) (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT of 0.4, 0.7 and 1.5 mg/kg/h IV, respectively. BMS-262084 increased ex vivo aPTT dose-dependently without changes in PT and TT. The antithrombotic effect of BMS-262084 was significantly correlated with its ex vivo aPTT, supporting the use of ex vivo aPTT as a pharmacodynamic biomarker. BMS-262084 did not alter ex vivo rabbit platelet aggregation to ADP and collagen. BT (fold-increase) determined at 3 and 10 mg/kg/h of BMS-262084 were 1.17 ± 0.04 and 1.52 ± 0.07*, respectively (*P < 0.05 vs. control). This study demonstrated that BMS-262084 prevented experimental thrombosis at doses with low BT effects in rabbits, and suggests that a small molecule FXIa inhibitor may represent a promising antithrombotic therapy.
Subject(s)
Azetidines/pharmacology , Factor XIa/antagonists & inhibitors , Fibrinolytic Agents/pharmacology , Piperazines/pharmacology , Platelet Activation/drug effects , Venous Thrombosis/drug therapy , Animals , Azetidines/adverse effects , Bleeding Time , Disease Models, Animal , Drug Evaluation, Preclinical , Fibrinolytic Agents/adverse effects , Male , Piperazines/adverse effects , Platelet Function Tests/methods , Rabbits , Venous Thrombosis/bloodABSTRACT
Apixaban has similar affinity for human and rabbit factor Xa (FXa). Rabbits are commonly used in development of thrombosis disease models; however, unlike in other species, apixaban demonstrated poor oral bioavailability (F = 3%) and a high clearance rate (2.55 l/h/kg) in rabbits. Oxidative metabolism of [14C] apixaban by liver microsomes was approximately 20 times faster in rabbits than in rats or humans. Following an intravenous (IV) dose of 5 mg/kg, circulating levels of [14C] apixaban decreased from the earliest sampling time (5 min) to undetectable at 4 h. After an oral dose of 30 mg/kg, levels of [14C] apixaban were only detected at 1 and 4 h. Radioactivity profiling showed that apixaban was a significant component in plasma only after IV administration; O-demethyl apixaban (M2), O-demethyl apixaban glucuronide (M14) and O-demethyl apixaban sulfate (M1) were prominent metabolites after both IV and oral administration. Studies of apixaban in rabbits showed a good correlation between apixaban concentrations and inhibition of FXa activity, prolongation of prothrombin time and modified prothrombin time, with no lag time between these ex vivo pharmacodynamic markers and plasma drug levels. The apixaban concentration required for 50% inhibition (IC50) of FXa activity ex vivo (0.22 +/- 0.02 microM) agreed with the IC50 from in vitro experiments in rabbit and human plasma. In summary, apixaban shows similar affinity for human and rabbit FXa. It produces a rapid onset of action, predictable concentration-dependent pharmacodynamic responses, and, unlike rats or humans, a rapid hepatic metabolism in rabbits.
Subject(s)
Anticoagulants/pharmacokinetics , Factor Xa Inhibitors , Pyrazoles/pharmacokinetics , Pyridones/pharmacokinetics , Administration, Oral , Animals , Anticoagulants/administration & dosage , Anticoagulants/metabolism , Blood Proteins/metabolism , Carbon Radioisotopes , Feces/chemistry , Female , Humans , Infusions, Intravenous , Male , Pyrazoles/administration & dosage , Pyrazoles/metabolism , Pyridones/administration & dosage , Pyridones/metabolism , RabbitsSubject(s)
Factor Xa Inhibitors , Fibrinolytic Agents/pharmacology , Pyrazoles/pharmacology , Pyridones/pharmacology , Thrombosis/blood , Administration, Oral , Dose-Response Relationship, Drug , Factor Xa/metabolism , Fibrinolytic Agents/administration & dosage , Humans , Peptide Fragments/blood , Prothrombin , Pyrazoles/administration & dosage , Pyridones/administration & dosageABSTRACT
The new P2Y(12) antagonist prasugrel produces greater inhibition of ADP-induced platelet aggregation (IPA) and reduction of thrombotic events in patients versus approved doses of clopidogrel, but increases major bleeding. We examined whether IPA level or P2Y(12) receptor occupancy (RO) could be optimized to better balance the efficacy and bleeding effects of these thienopyridines and reduce the response variability in rabbits. Rabbits were given three daily oral doses of clopidogrel (0.3-30 mg/kg/d), prasugrel (0.03-10 mg/kg/d) or vehicle (n = 6-40/group). Electrically-induced carotid artery thrombosis (AT, % thrombus weight reduction), cuticle bleeding time (BT, fold-increase over control), IPA to 20 microM ADP (% inhibition of peak light transmission) and RO (% inhibition of [(33)P]-2MeS-ADP binding to P2Y(1)-blocked platelets) were determined 2-3 hours after the last dose. ED(50) (doses for half-maximal effect, mg/kg/d) of AT, BT, IPA and RO were 1.6, 6.7, 1.9 and 1.4 for clopidogrel vs. 1.2, 1.9, 0.5 and 0.2 for prasugrel. IPA of 30-40% for both compounds produced the optimal balances of efficacy (AT: 50-60%) and BT of about 2-fold with significant RO of 70-80%. IPA of 50-60% achieved higher efficacy (AT: 60-80%), but with increased BT of five- to six-fold and >90% RO. Box-plot suggests no significant difference in the IPA and RO response variability between both compounds. Clopidogrel was 1.3-7 times less potent than prasugrel in rabbits, depending upon which biomarker was studied. The ratio of efficacy: bleeding was most favorable at a moderate IPA of 30% to 40%. Both compounds had similar IPA and RO response variability.
Subject(s)
Fibrinolytic Agents/pharmacology , Hemostasis/drug effects , Piperazines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Thiophenes/pharmacology , Thrombosis/drug therapy , Ticlopidine/analogs & derivatives , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/metabolism , Administration, Oral , Animals , Binding, Competitive , Bleeding Time , Clopidogrel , Disease Models, Animal , Dose-Response Relationship, Drug , Fibrinolytic Agents/administration & dosage , Fibrinolytic Agents/metabolism , Fibrinolytic Agents/toxicity , Hemorrhage/chemically induced , Male , Piperazines/administration & dosage , Piperazines/metabolism , Piperazines/toxicity , Platelet Aggregation Inhibitors/administration & dosage , Platelet Aggregation Inhibitors/metabolism , Platelet Aggregation Inhibitors/toxicity , Prasugrel Hydrochloride , Purinergic P2 Receptor Antagonists , Rabbits , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y12 , Thionucleotides/metabolism , Thiophenes/administration & dosage , Thiophenes/metabolism , Thiophenes/toxicity , Thrombosis/blood , Ticlopidine/administration & dosage , Ticlopidine/metabolism , Ticlopidine/pharmacology , Ticlopidine/toxicityABSTRACT
Target levels of ex vivo inhibition of platelet aggregation (IPA) induced by adenosine diphosphate (ADP) that produce clinically relevant effects of clopidogrel, a P2Y12 antagonist, are unclear. We examined standard and modified IPA and P2Y12 receptor occupancy as predictors of antithrombotic (% thrombus weight reduction) and bleeding time (BT, fold-increase over control) effects of clopidogrel in rabbit models of carotid artery thrombosis and cuticle bleeding, respectively. Standard and modified IPA with 20 microM ADP were measured in the absence and presence of partial P2Y1 blockade, respectively. Clopidogrel maximally produced standard IPA of 57% +/- 5%, antithrombotic effect of 85% +/- 1%, BT increase of 6.0 +/- 0.4-fold and P2Y12 receptor occupancy of 87% +/- 5%. Surprisingly, a clopidogrel dose that produced a low standard IPA of 17% +/- 4% and P2Y12 receptor occupancy of 39% +/- 5% achieved a significant antithrombotic activity of 55% +/- 2% with a moderate increase in BT of 2.0 +/- 0.1-fold. This underestimation of clopidogrel efficacy by standard IPA was improved by measuring either modified IPA or P2Y12 receptor occupancy. These results suggest that in clopidogrel-treated rabbits, low standard IPA is associated with significant antithrombotic effects. Moreover, modified IPA and P2Y12 receptor occupancy appear to better predict the magnitude of clopidogrel's efficacy compared with standard IPA, which may be a better predictor of BT.
Subject(s)
Blood Platelets/metabolism , Carotid Artery Thrombosis/prevention & control , Carotid Artery Thrombosis/physiopathology , Carotid Artery, Common/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Receptors, Purinergic P2/metabolism , Ticlopidine/analogs & derivatives , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/pharmacology , Analysis of Variance , Animals , Aspirin/pharmacology , Biomarkers/blood , Bleeding Time , Blood Platelets/drug effects , Carotid Artery Thrombosis/blood , Carotid Artery, Common/physiopathology , Clopidogrel , Disease Models, Animal , Dose-Response Relationship, Drug , Hemostasis/drug effects , Male , Predictive Value of Tests , Protein Binding/drug effects , Rabbits , Receptors, Purinergic P2/drug effects , Regional Blood Flow/drug effects , Thromboxane B2/blood , Ticlopidine/pharmacologyABSTRACT
Coactivation of platelets and the blood coagulation cascade contributes to the pathophysiology of arterial thrombosis. Combination therapy with antiplatelet and anticoagulant drugs may be needed for maximizing the prevention and treatment of arterial thrombosis. Few studies have thoroughly investigated the combined antithrombotic and bleeding effects of these antithrombotic agents. We, therefore, evaluated the antithrombotic and bleeding profiles of dual and triple therapy with razaxaban, a direct factor Xa inhibitor, plus aspirin and/or clopidogrel in rabbit models of electrolytic injury-induced carotid artery thrombosis and cuticle bleeding time, respectively. Compounds were infused either IV or into the portal vein from 1 h before arterial injury or cuticle transection to the end of experiment. Carotid blood flow was used as a marker of antithrombotic effect. We first evaluated the antithrombotic potency of razaxaban, and examined its ex vivo effects on coagulation parameters to confirm its selectivity. Antithrombotic ED(50) of razaxaban averaged 0.22 +/- 0.05 mg/kg/h (n = 6). Razaxaban at 3 mg/kg/h IV produced full antithrombotic efficacy, increased significantly ex vivo activated partial thromboplastin time and prothrombin time by 2.2 +/- 0.1- and 2.3 +/- 0.1-fold, respectively, and inhibited ex vivo factor Xa activity significantly by 91 +/- 5% (n = 6, P < 0.05) without affecting ex vivo thrombin activity. Razaxaban at concentrations up to 10 muM did not alter in vitro platelet aggregation responses to ADP, gamma-thrombin or collagen. To identify additive or synergistic antithrombotic effects of the various combination therapies, we purposefully used marginally effective doses of razaxaban at 0.1 mg/kg/h, aspirin at 0.3 mg/kg/h and clopidogrel at 1 mg/kg/h. Dual combination of threshold doses of razaxaban and aspirin or clopidogrel produced an enhanced antithrombotic effect without further increases in bleeding time. When compared with dual therapy with aspirin and clopidogrel (38 +/- 5% increase in blood flow), addition of razaxaban increased blood flow to 75 +/- 5% without additional bleeding time effects (n = 6/group, P < 0.05). In summary, razaxaban was an effective antithrombotic agent in a rabbit model of arterial thrombosis. Low-dose razaxaban was useful in combination with sub-optimal doses of aspirin and/or clopidogrel for the prevention of occlusive arterial thrombosis without excessive bleeding.
Subject(s)
Aspirin/therapeutic use , Factor Xa/pharmacology , Fibrinolytic Agents/pharmacology , Hemorrhage/physiopathology , Hemostasis , Isoxazoles/pharmacology , Pyrazoles/pharmacology , Ticlopidine/analogs & derivatives , Animals , Blood Coagulation/drug effects , Clopidogrel , Disease Models, Animal , Drug Therapy, Combination , Male , Platelet Aggregation/drug effects , Rabbits , Thrombosis/prevention & control , Ticlopidine/pharmacologyABSTRACT
DPC423 [1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide] is a synthetic, competitive, and selective inhibitor of coagulation factor Xa (fXa) (K(i): 0.15 nM in humans, 0.3 nM in rabbit). The objective of this study was to compare effects of DPC423, enoxaparin (low-molecular-weight heparin), and argatroban (thrombin inhibitor) on arterial thrombosis and hemostasis in rabbit models of electrically induced carotid artery thrombosis and cuticle bleeding, respectively. Compounds were infused i.v. continuously from 60 min before artery injury or cuticle transection to the end of experiment. Carotid blood flow was used as a marker of antithrombotic effect. Antithrombotic ED(50) values were 0.4 mg/kg/h for enoxaparin (n = 6), 0.13 mg/kg/h for argatroban (n = 6), and 0.6 mg/kg/h for DPC423 (n = 12). DPC423 at the maximum antithrombotic dose increased activated partial thromboplastin time and prothrombin time (n = 6) by 1.8 +/- 0.07- and 1.8 +/- 0.13-fold, respectively, without changes in thrombin time and ex vivo thrombin activity. The antithrombotic effect of DPC423 was significantly correlated with its ex vivo anti-fXa activity (r = 0.86). DPC423 at 1, 3, and 10 mg/kg p.o. increased carotid blood flow (percent control) at 45 min to 10 +/- 4, 24 +/- 6, and 74 +/- 7, respectively (n = 6/group). Cuticle bleeding times (percent change over control) determined at the maximum antithrombotic dose were 88 +/- 12 for argatroban, 69 +/- 13 for heparin, 4 +/- 3 for enoxaparin, 5 +/- 4 for DPC423, and -3 +/- 2 for the vehicle (n = 5-6/group), suggesting dissociation of antithrombotic and bleeding time effects for DPC423 and enoxaparin. The combination of aspirin and DPC423 at ineffective antithrombotic doses produced significant antithrombotic effect. Therefore, these results suggest that DPC423 is a clinically useful oral anticoagulant for the prevention of arterial thrombosis.