The article, "Factor Xa: Thrombokinase from Paul Morawitz to J Haskell Milstone" by Leonard M

Over the past few decades, drug discovery directed at the treatment and prevention of thromboembolic diseases has been challenged by the need to balance robust efficacy with improved safety relative to the standard of care. To this end, the most impactful advance to date has been the discovery and development of oral factor Xa inhibitors. In this essay, a brief account of the program that culminated in the discovery of Eliquis (apixaban) and the commitment to identify a compound with an optimal profile are described.

AimsThe 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.MethodsThis 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.ResultsBMS‐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).ConclusionBMS‐962212 has tolerability, PK and PD properties suitable for investigational use as an acute antithrombotic agent in Japanese or non‐Japanese subjects.

Objective— Warfarin is the current standard for oral anticoagulation therapy in patients with mechanical heart valves, yet optimal therapy to maximize anticoagulation and minimize bleeding complications requires routine coagulation monitoring, possible dietary restrictions, and drug interaction monitoring. As alternatives to warfarin, oral direct acting factor Xa inhibitors are currently approved for the prophylaxis and treatment of venous thromboembolism and reduction of stroke and systemic embolization. However, no in vivo preclinical or clinical studies have been performed directly comparing oral factor Xa inhibitors such as apixaban to warfarin, the current standard of therapy. Approach and Results— A well-documented heterotopic aortic valve porcine model was used to test the hypothesis that apixaban has comparable efficacy to warfarin for thromboprophylaxis of mechanical heart valves. Sixteen swine were implanted with a bileaflet mechanical aortic valve that bypassed the ligated descending thoracic aorta. Animals were randomized to 4 groups: control (no anticoagulation; n=4), apixaban oral 1 mg/kg twice a day (n=5), warfarin oral 0.04 to 0.08 mg/kg daily (international normalized ratio 2–3; n=3), and apixaban infusion (n=4). Postmortem valve thrombus was measured 30 days post-surgery for control-oral groups and 14 days post-surgery for the apixaban infusion group. Control thrombus weight (mean) was significantly different (1422.9 mg) compared with apixaban oral (357.5 mg), warfarin (247.1 mg), and apixiban 14-day infusion (61.1 mg; P<0.05). Conclusions— Apixaban is a promising candidate and may be a useful alternative to warfarin for thromboprophylaxis of mechanical heart valves. Unlike warfarin, no adverse bleeding events were observed in any apixaban groups.

In an effort to identify a potential back-up to apixaban (Eliquis®), we explored a series of diversified P4 moieties. Several analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent FXa binding affinity (FXa Ki), good human plasma anticoagulant activity (PT EC2x), cell permeability, and oral bioavailability.

The factor Xa inhibitor apixaban is one of the novel anticoagulants to emerge as alternatives to long-standing standards of care that include low-molecular-weight heparin and warfarin. The development of apixaban reflects a strategy to optimize the clinical pharmacology profile, dosing posology, trial designs, and statistical analyses across multiple indications, and to seek alignment with global health authorities. The primary objective of dose selection was to maintain balance between efficacy and bleeding risk. Twice-daily dosing of apixaban, rather than once daily, was chosen to lower peak concentrations and reduce fluctuations between peak and trough levels. Our discussion here focuses on the use of apixaban for stroke prevention in nonvalvular atrial fibrillation (NVAF). Supporting this indication, a pair of registrational trials was conducted that enrolled the full spectrum of patients who, by guidelines, were eligible for anticoagulation. In the AVERROES study of patients who were unsuitable for warfarin therapy, apixaban was superior to aspirin in reducing the risk of stroke or systemic embolism (SSE), without a significant increase in major bleeding (MB). In the ARISTOTLE (Apixaban for Reduction In STroke and Other ThromboemboLic Events in Atrial Fibrillation) study, apixaban was superior towarfarin on the rates of SSE, MB, and all-cause mortality. Overall, these studies have demonstrated a substantially favorable benefit-risk profile for apixaban over warfarin and aspirin in NVAF.

A 6-amidinotetrahydroquinoline screening hit was driven to a structurally novel, potent, and selective FVIIa inhibitor through a combination of library synthesis and rational design. An efficient gram-scale synthesis of the active enantiomer BMS-593214 was developed, which required significant optimization of the key Povarov annulation. Importantly, BMS-593214 showed antithrombotic efficacy in a rabbit arterial thrombosis model. A crystal structure of BMS-593214 bound to FVIIa highlights key contacts with Asp 189, Lys 192, and the S2 pocket.

Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P' binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.

Conventional prothrombin time (PT) assays have limited sensitivity and dynamic range in monitoring the anticoagulant activity of direct factor Xa inhibitors. Hence, new assays are needed. We modified a PT assay by adding calcium chloride (CaCl2) to the thromboplastin reagent to increase assay dynamic range and improve sensitivity. Effects of calcium and sodium ion concentrations, and sample handling, were evaluated to optimize assay performance. Increasing concentrations of calcium ions produced progressive increases in PT across the factor Xa inhibitor concentrations of 0 to 2500 nmol/L for razaxaban and apixaban. The greatest effect was seen when the thromboplastin reagent was diluted 1:2.25 with 100 mmol/L CaCl2 (thus selected for routine use). The optimized assay showed an interassay precision of 1.5 to 9.3 percentage coefficient of variation (%CV) for razaxaban and 3.1 to 4.6 %CV for apixaban. We conclude that the modified PT assay is likely to be suitable as a pharmacodynamic marker for activity at therapeutic concentrations of factor Xa inhibitors.

Inhibitors of the Tissue Factor/Factor VIIa (TF-FVIIa) complex are promising novel anticoagulants that show excellent efficacy and minimal bleeding in preclinical models. On the basis of a zwitterionic phenylglycine acylsulfonamide 1, a phenylglycine benzylamide 2 was shown to possess improved permeability and oral bioavailability. Optimization of the benzylamide, guided by X-ray crystallography, led to a potent TF-FVIIa inhibitor 18i with promising oral bioavailability, but promiscuous activity in an in vitro safety panel of receptors and enzymes. Introducing an acid on the pyrrolidine ring, guided by molecular modeling, resulted in highly potent, selective, and efficacious TF-FVIIa inhibitors with clean in vitro safety profile. The pyrrolidine acid 20 showed a moderate clearance, low volume of distribution, and a short t 1/2 in dog PK studies.

Bicyclic pyrazinone and pyrimidinone amides were designed and synthesized as potent TF–FVIIa inhibitors. SAR demonstrated that the S2 and S3 pockets of FVIIa prefer to bind small, lipophilic groups. An X-ray crystal structure of optimized compound 9b bound in the active site of FVIIa showed that the bicyclic scaffold provides 5 hydrogen bonding interactions in addition to projecting groups for interactions within the S1, S2 and S3 pockets. Compound 9b showed excellent FVIIa potency, good selectivity against FIXa, Xa, XIa and chymotrypsin, and good clotting activity.

Introduction: Factor Xa (FXa) is a critical enzyme in the coagulation cascade responsible for thrombin generation, the final enzyme that leads to fibrin clot formation. Significant success has recently been reported with compounds such as rivaroxaban, apixaban and edoxaban in the treatment and prevention of venous thromboembolism (VTE) and more recently in the prevention of stroke in atrial fibrillation (AF). The success these agents have demonstrated is now being reflected by a narrowing of new FXa patents over the past few years. The new patents appear to be structural modifications of previously published, small molecule inhibitors and bind in a similar manner to the FXa enzyme.Areas covered: SciFinder (R), PubMed and Google websites were used as the main source of literature retrieval. Patent searches were conducted in the patent databases: HCAPlus, WPIX and the full text databases (USPAT2, USPAT-FULL, EPFULL, PCTFULL) using the following keywords: ((FXa) OR (F OR factor) (W) (Xa)) (S) (inhibit? or block? or modulat? or antagonist? or regulat?). The search was restricted to patent documents with the entry date on or after 1 January 2009. Literature and information related to clinical development was retrieved from Thomson Reuter's Pharma.Expert opinion: A large body of Phase II and Phase III data is now available for FXa inhibitors such as rivaroxaban, apixaban, edoxaban and betrixaban. The clinical data demonstrate favorable benefit-risk profiles compared with the standards of care for short- and long-term anticoagulation (i.e., low molecular weight heparins (LMWHs) and wafarin). The potential exists that these agents will eventually be the agents of choice for the treatment of a host of cardiovascular disease states, offering improved efficacy, safety, and ease of use compared with existing anticoagulants.

Obesity and related sequelae such as insulin resistance and diabetes present a growing health care burden throughout the world. One of the hallmarks of the disease process in these patients is the presence of a prothrombotic state that manifests itself as heightened levels of circulating thrombotic/platelet biomarkers such as thrombin-antithrombin complex (T-AT), soluble CD40 ligand (sCD40L) and inflammatory cytokines such as interleukin 6 (IL6). We have found that levels of these biomarkers are also elevated in genetic models of obesity and diabetes (db/db mice). Apixaban is a potent, highly selective, direct Factor Xa inhibitor that shows great promise as a safer and more efficacious therapy then the existing standard of care (ARISTOTLE trial). For this reason, we sought to study the differences in the ability of apixaban and current clinical therapies to lower plasma markers of thrombosis (T-AT and sCD40L) in diabetic mice. We administered either apixaban, warfarin or aspirin and clopidogrel to 12 week old, male db/db mice p.o. for 48 h prior to collection of blood. T-AT and sCD40L were measured in plasma by ELISA. db/db mice had 3-fold greater levels of T-AT (P<0.05) and 2-fold (P<0.05) greater levels of sCD40L in plasma as compared to C57Bl6 mice. Warfarin (0.3 mg/kg) dose-dependently decreased T-AT (P<0.05) to levels similar to normoglycemic C57/Bl6 controls. Apixaban (5 and 50 mg/kg) significantly reduced (P<0.05) elevated levels of these biomarkers when administered b.i.d. When administered q.d., apixaban (10 and 100 mg/kg) lowered T-AT levels but did not robustly affect sCD40L. The platelet therapies, aspirin (30 mg/kg) and clopidogrel (3 mg/kg) robustly decreased sCD40L (P<0.05) but did not affect T-AT levels in obese, diabetic mice. Overall, these data suggest that administration of apixaban is equally efficacious as warfarin in lowering elevated thrombotic biomarkers in diabetic mice. Anti-platelet therapies were efficacious on platelet biomarkers but did not affect markers associated with elevated thrombin activation. This study was funded by Bristol-Myers Squibb and Pfizer.

To the Editor: In the Case Record, Cash and colleagues (Nov. 10 issue)1 report a case of limbic encephalitis with antibodies to leucine-rich, glioma-inactivated 1 (LGI1) in a 75-year-old man with amnesia and recurrent seizures. Some seizures described seem similar to “faciobrachial dystonic seizures.”2 Such seizures have characteristic signs and occur almost exclusively in association with LGI1 antibodies. Although seizure signs in the reported case were at times suggestive of more typical temporal-lobe epilepsy,3,4 some seizures were characterized by facial twitching, dysarthria, and hand posturing. These features are in keeping with the core distinctive features of faciobrachial dystonic seizures; namely, frequently recurring dystonic posturing of the arm, the face, or both. This posturing lasts only several seconds and interrupts motor tasks and speech, but without altered awareness.2 These episodes appear to be triggered by stress, an association also described by Irani and colleagues.2 When epileptic dystonia occurs in association with other forms of limbic encephalitis and other epileptic disorders, it is less prominent, it is of longer duration, it is associated with automatisms, and it spares the face.2-4 Some of the seizures described in this case were more consistent with faciobrachial dystonic seizures and hence predictive of the subsequent molecular diagnosis.