The effect of rivaroxaban on biomarkers in blood and plasma: a review of preclinical and clinical evidence.

Rivaroxaban is a direct, oral factor Xa inhibitor that is used for the prevention and treatment of various thromboembolic disorders. Several preclinical and clinical studies have utilized specific molecules as biomarkers to investigate the potential role of rivaroxaban beyond its anticoagulant activity and across a range of biological processes. The aim of this review is to summarize the existing evidence regarding the use of blood-based biomarkers to characterize the effects of rivaroxaban on coagulation and other pathways, including platelet activation, inflammation and endothelial effects. After a literature search using PubMed, almost 100 preclinical and clinical studies were identified that investigated the effects of rivaroxaban using molecular biomarkers. In agreement with the preclinical data, clinical studies reported a trend for reduction in the blood concentrations of D-dimers, thrombin-antithrombin complex and prothrombin fragment 1 + 2 following treatment with rivaroxaban in both healthy individuals and those with various chronic conditions. Preclinical and also some clinical studies have also reported a potential impact of rivaroxaban on the concentrations of platelet activation biomarkers (von Willebrand factor, P-selectin and thrombomodulin), endothelial activation biomarkers (matrix metalloproteinase-9, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) and inflammation biomarkers (interleukin-6, tumor necrosis factor-α and monocyte chemoattractant protein-1). Based on the results of biomarker studies, molecular biomarkers can be used in addition to traditional coagulation assays to increase the understanding of the anticoagulation effects of rivaroxaban. Moreover, there is preliminary evidence to suggest that rivaroxaban may have an impact on the biological pathways of platelet activation, endothelial activation and inflammation; however, owing to paucity of clinical data to investigate the trends reported in preclinical studies, further investigation is required to clarify these observations.

Effects of Rivaroxaban on Biomarkers of Coagulation and Inflammation: A Post Hoc Analysis of the X-VeRT Trial.

Introduction This X-VeRT (eXplore the efficacy and safety of once-daily oral riVaroxaban for the prevention of caRdiovascular events in patients with nonvalvular aTrial fibrillation scheduled for cardioversion) substudy evaluated the effects of treatment with rivaroxaban or a vitamin-K antagonist (VKA) on levels of biomarkers of coagulation (D-dimer, thrombin-antithrombin III complex [TAT] and prothrombin fragment [F1.2]) and inflammation (high sensitivity C-reactive protein [hs-CRP] and high-sensitivity interleukin-6 [hs-IL-6]) in patients with atrial fibrillation (AF) who were scheduled for cardioversion and had not received adequate anticoagulation at baseline (defined as, in the 21 days before randomization: no oral anticoagulant; international normalized ratio <2.0 with VKA treatment; or <80% compliance with non-VKA oral anticoagulant treatment). Methods Samples for biomarker analysis were taken at baseline ( n = 958) and treatment completion (42 days after cardioversion; n = 918). The influence of clinical characteristics on baseline biomarker levels and the effect of treatment on changes in biomarker levels were evaluated using linear and logistic models. Results Baseline levels of some biomarkers were significantly associated with type of AF (D-dimer and hs-IL-6) and with history of congestive heart failure (hs-CRP, D-dimer, and hs-IL-6). Rivaroxaban and VKA treatments were associated with reductions from baseline in levels of D-dimer (-32.3 and -37.6%, respectively), TAT (-28.0 and -23.1%, respectively), hs-CRP (-12.5 and -17.9%, respectively), and hs-IL-6 (-9.2 and -9.8%, respectively). F1.2 levels were reduced from baseline in patients receiving a VKA (-53.0%) but not in those receiving rivaroxaban (2.7%). Conclusion Anticoagulation with rivaroxaban reduced levels of key inflammation and coagulation biomarkers to a similar extent as VKAs, with the exception of F1.2. Further investigation to confirm the value of these biomarkers in patients with AF is merited.

BAY 1213790, a fully human IgG1 antibody targeting coagulation factor XIa: First evaluation of safety, pharmacodynamics, and pharmacokinetics.

BACKGROUND: Coagulation factor XI (FXI) contributes to the development of thrombosis but appears to play only a minor role in hemostasis and is therefore an attractive anticoagulant drug target. OBJECTIVES: To evaluate the safety, pharmacodynamic, and pharmacokinetic properties of BAY 1213790, a fully human immunoglobulin (Ig) G1 antibody targeting activated coagulation FXI (FXIa), in healthy men. METHODS: In this phase 1, single-blind, parallel-group, placebo-controlled, dose-escalation study, 83 healthy Caucasian men were randomized 4:1 to receive a single 60-minute intravenous infusion of BAY 1213790 (0.015-10 mg/kg) or placebo. Adverse events, pharmacodynamic parameters (including activated partial thromboplastin time [aPTT]) and pharmacokinetic parameters were determined. Volunteers were followed up for 150 days. RESULTS: BAY 1213790 demonstrated favorable safety and tolerability; there were no observed cases of bleeding or clinically relevant antidrug antibody formation. One volunteer (1.2%) experienced an infusion reaction. Following intravenous administration of BAY 1213790, dose-dependent increases in aPTT (maximal mean increase relative to baseline: 1.85 [conventional method] and 2.17 [kaolin-triggered method]) and rotational thromboelastometry whole blood clotting time were observed, as well as dose-dependent reductions in FXI activity. Bleeding times did not increase following administration of BAY 1213790 and were similar for all dose cohorts, including placebo. Measurable and dose-dependent increases in systemic exposure were detected for all doses of BAY 1213790 of 0.06 mg/kg or higher. CONCLUSIONS: Based on these safety, pharmacodynamic, and pharmacokinetic results, further evaluation of BAY 1213790 in patients with, or at risk of, thrombosis is warranted.

Targeted ex vivo reduction of CD64-positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B-based cytolytic fusion proteins.

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.

Granzyme M as a novel effector molecule for human cytolytic fusion proteins: CD64-specific cytotoxicity of Gm-H22(scFv) against leukemic cells.

Immunotoxins are promising targeted therapeutic agents comprising an antibody-based ligand that specifically binds to diseased cells, and a pro-apoptotic protein. Toxic components from bacteria or plants can trigger a neutralizing immune response, so that human effector molecules are more suitable. In this context, the protease granzyme B has been successfully tested in cytotoxicity assays against different cancer cells in vitro and in vivo. Our aim here was to introduce granzyme M as an alternative and novel component of human cytolytic fusion proteins. We fused it to the humanized single-chain antibody fragment (scFv) H22 which specifically binds to CD64, an FcγRI receptor overexpressed on activated myeloid cells and leukemic cells. We show that the humanized cytolytic fusion protein Gm-H22(scFv) specifically targets the acute myeloid leukemia cell line HL60 in vitro and is cytotoxic with an IC50 between 1.2 and 6.4 nM. These findings were confirmed ex vivo using leukemic primary cells from patients, which were killed by granzyme M despite the presence of the granzyme B inhibitor serpin B9. In conclusion, granzyme M is a promising new cell-death inducing component for hCFPs because it specifically and efficiently kills target cells when fused to a targeting component.

Design of human granzyme B variants resistant to serpin B9.

Human granzyme B (hGB) is a serine protease involved in immune-mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). Here, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine-scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild-type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild-type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications.