Supplementary Figure 5 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Materials, Figure Legends 1-6 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Tables 1-3 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Introduction Increasing use of factor Xa (FXa) inhibitors necessitates effective reversal agents to manage bleeding. Andexanet alfa, a novel modified recombinant human FXa, rapidly reverses the anticoagulation effects of direct and indirect FXa inhibitors. Objective To evaluate the ability of andexanet to reverse anticoagulation in vitro and reduce bleeding in rabbits administered edoxaban. Materials and methods In vitro studies characterized the interaction of andexanet with edoxaban and its ability to reverse edoxaban-mediated anti-FXa activity. In a rabbit model of surgically induced, acute hemorrhage, animals received edoxaban vehicle+andexanet vehicle (control), edoxaban (1 mg/kg)+andexanet vehicle, edoxaban+andexanet (75 mg, 5-minute infusion, 20 minutes after edoxaban), or edoxaban vehicle+andexanet prior to injury. Results Andexanet bound edoxaban with high affinity similar to FXa. Andexanet rapidly and dose-dependently reversed the effects of edoxaban on FXa activity and coagulation pharmacodynamic parameters in vitro. In edoxaban-anticoagulated rabbits, andexanet reduced anti-FXa activity by 82% (from 548±87 to 100±41 ng/ml; P<0.0001), mean unbound edoxaban plasma concentration by ~80% (from 100±10 to 21±6 ng/ml; P<0.0001), and blood loss by 80% vs. vehicle (adjusted for control, 2.6 vs. 12.9 g; P = 0.003). The reduction in blood loss correlated with the decrease in anti-FXa activity (r = 0.6993, P<0.0001) and unbound edoxaban (r = 0.5951, P = 0.0035). Conclusion These data demonstrate that andexanet rapidly reversed the anticoagulant effects of edoxaban, suggesting it could be clinically valuable for the management of acute and surgery-related bleeding. Correlation of blood loss with anti-FXa activity supports the use of anti-FXa activity as a biomarker for assessing anticoagulation reversal in clinical trials.

The objective of these studies was to compare 3-and 4-factor PCCs, approved for reversal of warfarin, with and exanet alfa in reversing anticoagulation effects of rivaroxaban, a direct FXa inhibitor, in a rabbit model of bleeding.

Event Abstract Back to Event Andexanet alfa for reversal of factor Xa inhibitors induced-anticoagulation in nonclinical and clinical studies Genmin Lu1, Polly Pine1, Janet Leeds1, Janice Castillo1, John Curnutte1 and Pamela Conley1 1 Portola Pharmaceuticals, Inc., United States Anticoagulants are widely used for the prevention and treatment of thrombosis but have the liability of bleeding due to over-anticoagulation. Traditional anticoagulants have an antidote for their reversal, such as vitamin K for reversal of warfarin and protamine for reversal of heparin. Although the newer classes of inhibitors targeting coagulation factor Xa (fXa) (direct fXa inhibitors and ATIII-dependent low molecular weight heparin (LMWH) and fondaparinux) have become the mainstay in thrombosis prevention and treatment, unlike heparin and warfarin, the newer anticoagulants lack an effective antidote to reverse the anticoagulant effects in case of urgent medical intervention or bleeding. Andexanet alfa (andexanet), an FDA-designated breakthrough therapy and orphan drug status currently in late stage clinical development, is a specific antidote for all fXa inhibitors. Andexanet is a recombinant modified human fXa that lacks the coagulation activity of native fXa but retains high binding affinity to all fXa inhibitors, thus sequestering the inhibitors and allowing for the restoration of normal hemostasis. Andexanet is expressed in Chinese hamster ovary (CHO) cells directly as a functional protein (not as a zymogen) and purified from harvested cell culture fluid without additional activation steps necessary for conversion of native fX to fXa. Nonclinical studies have demonstrated that andexanet binds to direct fXa inhibitors (rivaroxaban, apixaban, edoxaban, betrixaban) with high affinity (Kd = 0.5 -1.5 nM). It dose-dependently and completely reversed the anticoagulant activity of these inhibitors in buffered system with purified fXa as well as in human plasma as measured by anti-fXa activity and thrombin generation. In animal models of bleeding and blood loss (rats and rabbits), andexanet reduced anticoagulation-induced increase in blood loss by direct fXa inhibitors, such as rivaroxaban and edoxaban. Similarly, andexanet was also effective in reversal of ATIII-dependent fXa inhibitors (enoxaparin, fondaparinux) in both in vitro and in vivo animal models. Andexanet was well-tolerated in preclinical toxicology studies (rats and monkeys) and Phase 1 single ascending dose study in healthy volunteers. In Phase 2 studies in young healthy subjects, andexanet dose-dependently and significantly reversed pharmacodynamics markers of anticoagulation (anti-fXa, inhibition of thrombin generation) for all fXa inhibitors studied (apixaban, rivaroxaban, edoxaban and enoxaparin), and reduced the unbound inhibitor plasma concentrations for all direct fXa inhibitors. In Phase 3 randomized, double-blind, placebo controlled studies, older volunteers age 50 -70 were dosed with apixaban (5 mg twice daily) or rivaroxaban (20 mg daily) to steady state. Andexanet reversed the anticoagulant activity of apixaban and rivaroxaban within minutes following the IV bolus of andexanet and the reversal was maintained over the duration of infusion for 2 hours and was well tolerated. The studies met the primary and all secondary endpoints with high statistical significance. In conclusion, andexanet is a specific, rapidly-acting antidote being developed for urgent reversal of fXa inhibitor anticoagulant activity. The ongoing Phase 3b/4 study is evaluating the efficacy and safety of andexanet in patients with fXa inhibitor-associated acute major bleeding. Keywords: in vivo, in vitro, Clinical Trial, protein Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Biomaterials in thrombosis and hemostasis Citation: Lu G, Pine P, Leeds J, Castillo J, Curnutte J and Conley P (2016). Andexanet alfa for reversal of factor Xa inhibitors induced-anticoagulation in nonclinical and clinical studies. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.03004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Genmin Lu Polly Pine Janet Leeds Janice Castillo John Curnutte Pamela Conley Google Genmin Lu Polly Pine Janet Leeds Janice Castillo John Curnutte Pamela Conley Google Scholar Genmin Lu Polly Pine Janet Leeds Janice Castillo John Curnutte Pamela Conley PubMed Genmin Lu Polly Pine Janet Leeds Janice Castillo John Curnutte Pamela Conley Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Introduction: Andexanet alfa (andexanet) is a modified recombinant human factor Xa (FXa), developed to reverse the anticoagulation effects of both direct and indirect FXa inhibitors. For antithrombin III (ATIII)-dependent FXa inhibitors, such as enoxaparin, andexanet binds to the ATIII-enoxaparin complex with high affinity and reverses the inhibition of coagulation factors Xa and IIa. This study evaluated the ability of andexanet to reverse anticoagulation effects and blood loss due to the indirect FXa inhibitor, enoxaparin, in a rabbit liver laceration model.

Background: Janus kinases (JAKs) are regulators of signaling through cytokine receptors. The importance of JAK1/3 signaling on T(H)2 differentiation and development of lung allergic responses has not been investigated.Objective: We sought to examine a selective JAK1/3 inhibitor (R256) on differentiation of T-H subsets in vitro and on development of ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) and inflammation in an experimental model of asthma.Methods: A selective JAK1/3 inhibitor was used to assay the importance of this pathway on induction of T(H)1, T(H)2, and T(H)17 differentiation in vitro. In vivo, the effects of inhibiting JAK1/3 signaling were examined by administering the inhibitor during the sensitization or allergen challenge phases in the primary challenge model or just before provocative challenge in the secondary challenge model. Airway inflammation and AHR were examined after the last airway challenge.Results: In vitro, R256 inhibited differentiation of T(H)2 but not T(H)1 or T(H)17 cells, which was associated with downregulation of signal transducer and activator of transcription (STAT) 6 and STAT5 phosphorylation. However, once polarized, T(H)2 cells were unaffected by the inhibitor. In vivo, R256 administered during the OVA sensitization phase prevented the development of AHR, airway eosinophilia, mucus hypersecretion, and T(H)2 cytokine production without changes in T(H)1 and T(H)17 cytokine levels, indicating that selective blockade of T(H)2 differentiation was critical. Inhibitor administration after OVA sensitization but during the challenge phases in the primary or secondary challenge models similarly suppressed AHR, airway eosinophilia, and mucus hypersecretion without any reduction in T(H)2 cytokine production, suggesting the inhibitory effects were downstream of T(H)2 cytokine receptor signaling pathways.Conclusions: Targeting the T(H)2-dependent JAK/STAT activation pathway represents a novel therapeutic approach for the treatment of asthma.

WRM 130 Survey of Analytical Techniques Useful for Thin Film Material Evaluation in High Technology Applications Hugh E. Gotts, hugh.gotts@airliquide.com.Balazs NanoAnalysis, Air Liquide Electronics, Fremont, CA 94538, United States The pervasive use of organic and inorganic materials to produce thin films in semiconductor, MEMS, Flat panel, and PV industries have challenged the sensitivity and specificity of modern analytical and physical chemical techniques. The necessity to continuously produce films of a consistent thickness, purity, and morphology requires analytical tools which are capable of measuring the required parameters quickly and accurately. As this symposium will concentrate on the application of a set of analytical methodologies, this talk will provide a guide to the techniques used. It is important to characterize the composition of organo-metallic precursors via a gas chromatographic technique (GC-MS or TC-TCD/FID). Non-volatile thermally labile materials are typically analyzed using a liquid chromatographic approach (LC-MS). Trace metal (TM) content in precursor materials are accurately determined using a variety of Inductively Coupled PlasmaMass Spectroscopy (ICP-MS) techniques. Once the thin film has been produced, TM measurements are performed utilizing LA-ICP-MS, dynamic SIMS or TOF-SIMS. Morphology is determined utilizing AFM, SEM or TEM techniques. Other techniques will be discussed as appropriate.

Abstract JAK are major downstream regulators of signaling through cytokine receptors, activating STAT. Effective targeting of the Th2-associated JAK-STAT pathway would represent a new therapeutic approach for the treatment of asthma. Here, we studied the effects of a novel selective JAK1/3 inhibitor, R256, on Th1, Th2, and Th17 differentiation in vitro and on the development of ovalubmin-induced AHR and airway inflammation in vivo in mice. R256 prevented only Th2 differentiation in vitro and was associated with the downregulation of STAT5 and STAT6 phosphorylation. In contrast, Th2 cells were unaffected by the inhibitor when added after polarization was complete. In vivo, R256 administered during the allergen sensitization phase prevented the development of AHR, airway eosinophilia, mucus hypersecretion, and Th2 cytokine production without altering Th1 and Th17 cytokine production, indicating that selective blockade of Th2 differentiation was critical. Inhibitor administration after allergen sensitization but during the challenge phase also suppressed AHR, airway eosinophilia, and mucus hypersecretion but no effect on Th2 cytokine levels in BAL could be detected, suggesting effects on downstream cytokine receptor signaling pathways. Thus, R256-mediated inhibition of JAK1/3 activation was effective when administered both prior to and after Th2 polarization to attenuate allergen-induced Th2 dominant airway inflammation and AHR.

Tissue injury following ischemia-reperfusion (I/R) occurs as a consequence of actions of soluble factors and immune cells. Growing evidence supports a role for platelets in the manifestation of tissue damage following I/R. Spleen tyrosine kinase has been well documented to be important in lymphocyte activation and more recently in platelet activation. We performed experiments to evaluate whether inhibition of platelet activation through inhibition of spleen tyrosine kinase prevents tissue damage after mesenteric I/R injury. Platelets isolated from C57BL/6J mice fed with R788 for 10 days were transfused into C57BL/6J mice depleted of platelets 2 days before mesenteric I/R injury. Platelet-depleted mice transfused with platelets from R788-treated mice before mesenteric I/R displayed a significant reduction in the degree of remote lung damage, but with little change in the degree of local intestinal damage compared with control I/R mice. Transfusion of R788-treated platelets also decreased platelet sequestration, C3 deposition, and immunoglobulin deposition in lung, but not in the intestine, compared with control groups. These findings demonstrate that platelet activation is a requisite for sequestration in the pulmonary vasculature to mediate remote tissue injury after mesenteric I/R. The use of small-molecule inhibitors may be valuable to prevent tissue damage in remote organs following I/R injury.

Reperfusion injury to tissue following an ischemic event occurs as a consequence of an acute inflammatory response that can cause significant morbidity and mortality. Components of both the innate (complement, immunoglobulin, monocytes, and neutrophils) and adaptive (B and T lymphocytes) immune systems have been demonstrated to mediate tissue injury. Spleen tyrosine kinase (Syk) is responsible for membrane-mediated signaling in various cell types including B lymphocytes, macrophages, and T cells. We investigated the ability of a small drug Syk inhibitor, R788, to protect mice against mesenteric ischemia-reperfusion (I/R)-induced local (intestine) and remote lung injury. Mice were fed with chow containing a Syk inhibitor for 6 days before the performance of intestinal I/R, which resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local and remote lung injury. The beneficial effect was associated with reduced IgM and complement 3 deposition in the tissues and significant reduction of polymorphonuclear cell infiltration. Our data place Syk upstream of events leading to the binding of natural antibodies to the ischemia-conditioned tissues and urge the consideration of the use of Syk inhibitors in the prevention or improvement of tissue injury of organs exposed to ischemia or hypoperfusion.

The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.

OBJECTIVE:Spleen tyrosine kinase (Syk) is involved in membrane-mediated signaling in various cells, including immune cells. It is overexpressed in T cells from patients with systemic lupus erythematosus (SLE), and its inhibition has been shown to improve T cell function as well as to improve disease manifestations in (NZB x NZW)F(1) lupus-prone mice and in patients with rheumatoid arthritis. While clinical trials examining Syk inhibition in patients with SLE are being considered, the aim of our experiments was to determine whether the therapeutic effects of Syk inhibition extend to other strains of lupus-prone mice and whether they result in improvement in skin disease and modification of established disease. METHODS:Female MRL/lpr or BAK/BAX mice were studied. Starting either at age 4 weeks (before disease) or at age 16 weeks (after established disease) and continuing for up to 16 weeks, mice were fed chow containing the Syk inhibitor R788 or control chow. RESULTS:We found that inhibition of Syk in MRL/lpr and BAK/BAX mice prevented the development of skin disease and significantly reduced established skin disease. Similarly, Syk inhibition reduced the size of the spleen and lymph nodes, suppressed the development of renal disease, and suppressed established renal disease. Discontinuation of treatment resulted in extended suppression of skin disease for at least 8 weeks and suppression of renal disease for 4 weeks. CONCLUSION:Syk inhibition suppresses the development of lupus skin and kidney disease in lupus-prone mice, suppresses established disease in lupus-prone mice, and may represent a valuable treatment for patients with SLE.