scholarly journals Elucidating the Excessive Pro-Coagulant Effect of a Sequence Identical Analogue to ACE910 in Combination with Bypassing Agents

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 90-90
Author(s):  
Rudolf Hartmann ◽  
Tjerk Feenstra ◽  
Sabine Knappe ◽  
Michael Dockal ◽  
Friedrich Scheiflinger
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Thrombus Formation ◽  
Phase Iii ◽  
Hek293 Cells ◽  
Employment Equity ◽  
Equity Ownership ◽  
Bypassing Agents

Abstract Introduction: Emicizumab (ACE910), an antibody to FIX(a) and FX(a), is currently under investigation for treatment of hemophilia with inhibitors. In a phase III trial, two thromboembolic complications and three cases of microangiopathy were reported in patients on ACE910 prophylaxis [Oldenburg et al. NEJM 2017], whose breakthrough bleeding was treated with activated prothrombin complex concentrate aPCC (FEIBA) or aPCC and rFVIIa. We generated a sequence identical analogue (SIA) to ACE910 and analyzed its synergistic interplay with bypassing agents. Aims: To monitor in vitro the pro-coagulant activity of SIA ACE910 in the presence of FEIBA and rFVIIa, and detect the source of excessive coagulation induced by SIA ACE910 combined with FEIBA. Methods: A sequence identical analogue (SIA) to ACE910 was expressed in HEK293 cells, purified as previously described [Sampei et al. PLoS One 2013], and analyzed in several global hemostatic assays at different concentrations and test conditions using plasma and whole blood assays. In thrombin generation (TG) experiments, platelet-poor plasma (PPP) from hemophilia A inhibitor patients and hemophilia A plasma reconstituted with platelets from 3 healthy donors (PRP) was used. A normal TG range was established in healthy donor plasma. Therapeutic concentrations of SIA ACE910 (20-600 nM) were tested alone and with FEIBA (0.05-1 U/mL) or rFVIIa (0.88-5.25 µg/mL). To measure FEIBA components' contribution to the synergistic effect with SIA ACE910, PPP was spiked with select FEIBA components at concentrations corresponding to 0.5 U/mL FEIBA in combination with the antibody. Thrombus formation was analyzed in FVIII-inhibited blood using rotational thromboelastometry (ROTEM) and Total Thrombus-formation Analysis System (T-TAS). Results: Normal peak thrombin was 47-144 nM for PPP and 88-231 nM for PRP. rFVIIa and FEIBA had an additive effect on TG in combination with SIA ACE910 in both plasma types. Combined with rFVIIa (0.88 µg/mL) or FEIBA (0.5 U/mL), SIA ACE910 (600 nM) induced a ~2- and ~16-fold increase over SIA ACE910 alone. SIA ACE910+rFVIIa did not reach the normal range, while SIA ACE910+FEIBA far exceeded it. Adding individual FEIBA components to PPP showed that FIX was, with a half-maximal effect, the main driver for enhanced TG, followed by FIXa. formation in FVIII-inhibited whole blood using ROTEM and T-TAS confirmed the excessive effect of SIA ACE910+FEIBA. In ROTEM, FEIBA and rFVIIa reduced clotting time to shorter than normal, whereas SIA ACE910 had only little effect. Moreover, adding SIA ACE910 to rFVIIa exerted no effect over rFVIIa alone. Conclusion: Combining SIA ACE910 at plasma concentrations observed in patients [Oldenburg et al. NEJM 2017] with FEIBA induced excessive thrombin generation and faster clot formation. In vitro, this effect is mainly mediated by FEIBA component FIX. ACE910 binds to FIX and FIXa to the same extent, and displays its pro-coagulant effect via an unregulated mechanism. Therefore, careful judgement is needed in treating breakthrough bleeds with FEIBA. Disclosures Hartmann: Shire: Employment. Feenstra: Shire: Employment. Knappe: Shire: Employment. Dockal: Baxalta: Patents & Royalties; Shire: Employment, Equity Ownership; Baxter: Equity Ownership, Patents & Royalties. Scheiflinger: Baxter: Equity Ownership; Shire: Employment, Equity Ownership.

scholarly journals Thrombin Generation Assay in Plasma from Hemophilia a Patients with or without Inhibitors on Concizumab Prophylaxis: Spiking with rFVIIa or aPCC

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3639-3639
Author(s):  
Marianne Kjalke ◽  
Mads Kjelgaard-Hansen ◽  
Søren Andersen ◽  
Ida Hilden
Keyword(s):  
Thrombin Generation ◽  
Significant Contribution ◽  
Hemophilia A ◽  
Employment Equity ◽  
Breakthrough Bleeding ◽  
Thrombin Generation Assay ◽  
Bleeding Episodes ◽  
Equity Ownership ◽  
Bypassing Agents

Introduction: Concizumab is a humanized monoclonal antibody that inhibits tissue factor pathway inhibitor (TFPI). Concizumab is currently in clinical development as a subcutaneous prophylactic therapy for hemophilia A and B patients with and without inhibitors. Breakthrough bleeding episodes experienced by inhibitor patients while on concizumab prophylaxis may be treated with the bypassing agents recombinant activated factor VII (rFVIIa; NovoSeven®) or activated prothrombin complex concentrate (aPCC; FEIBA®). Aim: To investigate the in vitro effect of rFVIIa and aPCC on hemophilia A plasma containing concizumab using a thrombin generation assay and pooled plasma spiked with concizumab or samples from patients treated prophylactically with concizumab. Methods: Pooled hemophilia A plasma was spiked with concizumab at 1, 3 and 10 nM and patient plasma samples from explorer4 (n=16; hemophilia with inhibitors; NCT03196284) and explorer5 (n=30; hemophilia A; NCT03196297) before and during concizumab prophylaxis at steady state exposure levels were assessed. Samples were spiked with rFVIIa (25 or 75 nM) or aPCC (0.25, 0.5 or 1 U/mL), and analyzed using a thrombin generation assay initiated with tissue factor (PPP-Low, Thrombinoscope). The effects of rFVIIa or aPCC in the absence or presence of concizumab were compared using ANOVA methodology. Results: Addition of rFVIIa or aPCC to hemophilia A plasma with or without inhibitors increased peak thrombin generation both in the absence and presence of concizumab. A significant additional effect of rFVIIa and aPCC was observed for all concizumab concentrations spiked to the plasma pool. Overall, the effects of the combination of concizumab and rFVIIa or aPCC were mainly additive; however, a small but statistically significant drug-drug interaction was observed for rFVIIa (25 nM or 75 nM) and aPCC (0.5 U/ml or 1 U/mL) in the presence of 10 nM concizumab. At this concizumab concentration, the additive effect of aPCC corresponded to 68% of the total observed effect and the additive effect of rFVIIa to 85% of the total observed effect. At lower concizumab concentrations (1 and 3 nM), statistically significant drug-drug effects were only observed in combination with aPCC. No excessive thrombin generation above the level obtained with 1 IU/mL recombinant factor VIII (rFVIII) was observed at 1 nM concizumab combined with either rFVIIa (25 and 75 nM) or aPCC 0.5 U/mL. However, addition of 1 U/mL aPCC to 1 nM concizumab resulted in a thrombin peak modestly above the upper 95% confidence interval of the rFVIII range. In the experiments using plasma from patients treated with concizumab, the increase in thrombin peak upon addition of rFVIIa was within or below the range observed by spiking with 1 IU/mL rFVIII. The increase in thrombin peak upon addition of aPCC was within or above the rFVIII range. The effects of concizumab and rFVIIa or aPCC were mainly additive; however, a small, statistically significant contribution caused by drug-drug interaction was observed for concizumab and rFVIIa (75 nM) in both plasma from patients with and without inhibitors, and for 1 U/mL aPCC in plasma from patients with inhibitors. The additive effects of concizumab and rFVIIa corresponded to between 60% (25 nM rFVIIa, plasma without inhibitors) and 75% (75 nM rFVIIa, inhibitor plasma), and the additive effects of concizumab and 1 U/mL aPCC corresponded to 77% of the total observed effects. Conclusions: Addition of rFVIIa or aPCC to hemophilia A plasma with or without inhibitors increased peak thrombin generation as expected both in the absence and presence of concizumab. Thus, the bypassing agents function as expected in plasma containing concizumab. The effects of concizumab and rFVIIa or aPCC were mainly additive. A small but statistically significant contribution was synergistic in accordance with the concizumab mechanism of action (Hilden I et al, Blood, 2012). These in vitro results support the concomitant use of bypassing agents to treat breakthrough bleeding episodes in hemophilia with inhibitor patients on concizumab prophylactic treatment. Disclosures Kjalke: Novo Nordisk A/S: Employment, Honoraria. Kjelgaard-Hansen:Novo Nordisk A/S: Employment, Equity Ownership. Andersen:Novo Nordisk A/S: Employment, Equity Ownership, Honoraria. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties.

Antithrombin Reduction Corrected Thrombin Generation in Samples from Hemophilia A and B Patients with Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 552-552 ◽  
Author(s):  
Gili Kenet ◽  
Tami Livnat ◽  
Emma Fosbury ◽  
Pratima Chowdary ◽  
Alfica Sehgal ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Severe Hemophilia ◽  
Employment Equity ◽  
Advisory Committees ◽  
Advisory Boards ◽  
Equity Ownership

Abstract Background: Severe hemophilia A and B patients with inhibitors experience serious musculoskeletal hemorrhage as well as high risk of limb and life threatening bleeds. However, lack of effect of FVIII or FIX substitution therapy and short functional half-life of by-passing agents, leave these patients with very limited bleed preventive treatment options. ALN-AT3 (Alnylam Pharmaceuticals, Cambridge, MA, USA), a subcutaneously administered investigational RNAi therapeutic targeting reduction of antithrombin for potential treatment of hemophilia is currently in phase 1 clinical development in hemophilia A and B patients without inhibitors. Initial data from that ongoing study in 12 patients suggest an AT KD dependent correction of thrombin generation. This study aims to assess changes in peak thrombin generation in samples from patients with severe hemophilia A and B with inhibitors following in vitro reduction of antithrombin. Materials and methods: Citrated plasma samples were obtained from patients with severe hemophilia A and B with high responding inhibitors. Samples were spiked in vitro with isotype specific control IgG or a monoclonal antibody (Haemtech Inc, Essex Junction, VT, USA) targeting antithrombin knockdown of 50% and 90%. Dynamic formation of thrombin was measured by calibrated automated thrombin generation using 1pM tissue factor PPP reagent and 4μM phospholipid (Thrombinoscope, Maastricht, The Nederlands). The primary effect measure was peak thrombin (nM). Data were tested by a 1-way ANOVA and p<0.05 was considered statistically significant. Results: A total of 12 inhibitor hemophilia samples were investigated; 9 hemophilia A and 3 hemophilia B. All the control samples demonstrated a profound defect in thrombin generation with a median peak thrombin of 19.9 nM (range 6.7 - 42.4). Patients with severe hemophilia A and inhibitors had a median peak thrombin generation of 19.7 nM (range 6.7 - 42.4), whereas patients with severe hemophilia B and inhibitors had a median peak thrombin generation of 19.2nM (range 19.4 - 38.1). An AT reduction dependent improvement in peak thrombin generation was observed in all 12 tested plasma samples (Figure 1). In the first 12 subjects, peak thrombin generation was increased up to 363% from a mean of 22nM (control) to 39 nM (50% AT reduction) and 80nM (90% AT reduction) (p<0.05); levels comparable to thrombin generation observed in healthy male volunteers and in hemophilia patients treated with ALN-AT3. Conclusions: These in vitro data suggest that reduction of AT is a promising approach for restoring hemostatic balance and correcting thrombin generation in hemophilia patients with inhibitors. Furthermore, the present laboratory data compare well with clinical data generated with ALN-AT3 administered to patients with hemophilia A or B. Thus, both laboratory and emerging clinical data suggest that targeting antithrombin could be a promising approach for restoring hemostatic balance in hemophilia. The potential for low volume subcutaneous administration, infrequent dosing, and applicability to persons with hemophilia who have inhibitors, make ALN-AT3 a particularly encouraging investigational therapy. Figure 1. Figure 1. Disclosures Kenet: Bayer, Novo Nordisk: Other: Advisory Boards, Speakers Bureau; Opko Biologics: Consultancy, Other: Advisory Boards; BPL; Baxelta: Research Funding; Pfizer: Honoraria. Off Label Use: ALN-AT3 is an investigational RNAi therapeutic targeting the endogenous anticoagulant antithrombin.. Chowdary:Sobi: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees. Sehgal:Alnylam Pharmaceuticals: Employment, Equity Ownership. Akinc:Alnylam Pharmaceuticals: Employment, Equity Ownership. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.

scholarly journals Mim8 - a Next-Generation FVIII Mimetic Bi-Specific Antibody - Potently Restores the Hemostatic Capacity in Hemophilia a Settings in Vitro and In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 96-96 ◽  
Author(s):  
Stine L Kjellev ◽  
Henrik Østergaard ◽  
Per Jr Greisen ◽  
Mette B Hermit ◽  
Karina Thorn ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Bispecific Antibody ◽  
Hemophilia A ◽  
Next Generation ◽  
Employment Equity ◽  
Thrombin Generation Assay ◽  
Equity Ownership ◽  
Target Binding

The treatment of hemophilia A (HA) is primarily based on replacement of factor VIII (FVIII), and in people with HA with inhibitors (HAwI) on the use of by-passing agents. Recently, a FVIII mimetic bispecific antibody emicizumab (Hemlibra®) was approved for treatment of HA and HAwI, offering a subcutaneous, prophylactic treatment opportunity with potential for significantly reducing the treatment burden. We describe the development and pre-clinical characterization of Mim8, a novel, next-generation FVIII mimetic human bispecific antibody. Mim8 is a highly potent molecule bridging factor IXa (FIXa) and factor X (FX) in development for subcutaneous treatment of people with HA and HAwI. Development of Mim8 utilized the Duobody® platform to initially screen for compatible anti-FIXa and anti-FX antibodies followed by several iterations of systematic mutational optimization. In total, more than 30,000 bispecific antibodies were analyzed. The optimization process aimed for efficient Mim8-mediated activation of FX by FIXa in the presence of procoagulant membrane, low target binding in solution, low immunogenicity risk, and for desirable biophysical parameters such as low viscosity. In vitro characterization demonstrated that Mim8 efficiently localizes FIXa and FX to the phospholipid surface and enhances FXa activation. The monovalent anti-FIXa arm alone stimulates the proteolytic activity of FIXa in the range of 15,000-fold and is an important contributor to the activity of the bispecific antibody. The dissociation constants (Kd) of Mim8 for FIXa and FX is in the micromolar range, minimizing target binding in the blood. Using thrombin generation assay in congenital HA plasma and thrombelastography (TEG) in whole blood from healthy volunteers spiked with anti-FVIII antibodies, Mim8 was capable of normalizing thrombin generation and blood clot formation, respectively, with approximately 15 times greater potency than emicizumab (Figure 1). A similar potency improvement was demonstrated in a tail vein transection bleeding model in FVIII-deficient mice co-dosed with human FIX and FX to circumvent lack of Mim8 cross reactivity to murine FIX and FX. The terminal half-life of Mim8 was estimated to 14 days (range 10-17 days) in cynomolgus monkeys and the subcutaneous bioavailability to 97%. In conclusion, Mim8 is a novel, next-generation FVIII mimetic bispecific antibody with anti-FIXa and anti-FX arms that potently stimulates FX activation resulting in efficacious haemostasis in vitro and in vivo. Mim8 has a high potency allowing for administration of small volumes in a pen device, good PK parameters, minimal target binding in the blood, and good biophysical properties. Collectively, these properties support clinical development of Mim8 as a potentially improved next-generation FVIII-mimetic prophylactic treatment option for persons with hemophilia A regardless of inhibitor status. Figure 1: Left: FXI-triggered thrombin generation assay in congenital HA plasma (mean and SD of n = 5). Right: thromboelastography in whole blood from healthy donors spiked with polyclonal anti-FVIII antibody (mean and SD of n = 3). Coagulation was triggered with low concentration (∼30 fM) of tissue factor (Innovin® 1:200,000). Shaded areas: standard deviation of controls. Blue circles: Mim8. Grey squares: a sequence identical analogue (SIA) to emicizumab (comparable data were obtained with a commercially available batch of Hemlibra®). Disclosures Kjellev: Novo Nordisk A/S: Employment, Equity Ownership. Østergaard:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Greisen:Novo Nordisk A/S: Equity Ownership, Patents & Royalties: Patents. Hermit:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Thorn:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hansen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Zhou:Novo Nordisk A/S: Equity Ownership, Other: Previous employment, Patents & Royalties: Patents. Bjelke:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Kjalke:Novo Nordisk A/S: Employment, Honoraria. Lund:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Holm:Novo Nordisk A/S: Equity Ownership, Other: Previous employment. Ley:Novo Nordisk A/S: Employment, Equity Ownership. Elenius:Novo Nordisk A/S: Equity Ownership, Other: Previous employment; Leo Pharma A/S: Employment, Equity Ownership. Thygesen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Loftager:Novo Nordisk A/S: Employment, Equity Ownership. Rasch:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lorenzen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Gandhi:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lamberth:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Egebjerg:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lund:Novo Nordisk A/S: Employment, Equity Ownership. Henriksen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Rahbek-Nielsen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Yang:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Phospholipid-Independent Activity of Fviiia Mimetic Bispecific Antibodies in Plasma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2461-2461
Author(s):  
Maria M Aleman ◽  
Siddharth Jindal ◽  
Nina Leksa ◽  
Robert Peters ◽  
Joe Salas
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Bispecific Antibodies ◽  
Molar Ratio ◽  
Phospholipid Vesicles ◽  
Employment Equity ◽  
One Stage ◽  
Equity Ownership ◽  
Substrate Cleavage

Abstract Introduction: An important coagulation regulatory mechanism is localization of clotting complexes to exposed phosphatidylserine (PS) on cell surfaces. All components of the intrinsic tenase complex (factor (F)VIIIa, FIXa, and FX) bind to PS. FVIIIa mimetic bispecific antibodies are drugs in development for hemophilia A that aim to mimic the cofactor function of FVIIIa by bringing together FIXa and FX to generate FXa. However, these antibodies differ from FVIII in many ways including no requirement of activation and a lack of direct PS binding. Emicizumab is a bispecific antibody currently on the market for hemophilia A patients with inhibitors. It binds to factor FIX, FIXa, FX, and FXa with micromolar affinities in solution. Previously, we have shown that in-house preparations of sequence-identical emicizumab (SI-Emi) showed similar weak affinities to its antigens and similar in vitro activity to published emicizumab results by one-stage clotting, chromogenic FXa generation, and thrombin generation. However, in chromogenic FXa generation using antibody concentrations in the range of the mean steady state plasma concentration of patients on emicizumab prophylaxis [~360 nM, (Oldenburg, et al., NEJM 2017)], SI-Emi maintained 28% of its activity even in the absence of PS-containing phospholipid vesicles. Another FVIIIa mimetic antibody, BS-027125, was discovered by our group and binds with low nanomolar affinity to FIX, FIXa and FX, with no detectable binding to FXa. In one-stage clotting, BS-027125 achieved clot times similar to physiological levels of FVIII, but had poor activity in thrombin generation at these concentrations. Furthermore, it too maintained small amounts of phospholipid-independent activity in chromogenic FXa generation. Given the artificial nature of the chromogenic FXa generation assay, and that activity of prothrombinase is PS-dependent thereby precluding omission of phospholipids from thrombin generation assays, we developed an assay to detect FXa generation in a plasma milieu by FVIIIa mimetic antibodies or FVIII with and without phospholipid vesicles. Methods: FVIIIa mimetic antibodies or recombinant FVIII (rFVIII) were incubated with thrombin for 5 minutes, quenched with hirudin, then spiked into platelet-free congenital hemophilia A plasma treated with additional hirudin. FXIa (to generate FIXa in situ) with and without PC:PE:PS (40:40:20 molar ratio) phospholipid vesicles was added and reactions were triggered with a solution of CaCl2 and fluorogenic FXa substrate (Mes-D-LGR-ANSN(C2H5)2). Substrate cleavage was monitored kinetically on a fluorescent plate reader. Substrate cleavage by FXIa could not be detected, yet another unknown plasma peptidase did cleave substrate at a constant low rate that was background subtracted. Results: In the absence of phospholipid vesicles, SI-Emi maintained 51±3.7% of its FXa generation activity at all concentrations tested (3.8±0.4 versus 8.0±1.1 RFU/min at 333 nM). BS-027125 showed very low activity (0.43±0.12 RFU/min at 50 nM) in the presence of phospholipid vesicles, however, in the absence of phospholipid vesicles, BS-027125 activity was not detectable above baseline. Nearly all rFVIII activity (>99%) was lost in the absence of phospholipid vesicles (0.14±0.04 versus 15.1±1.8 RFU/min at 0.3 IU/mL). Addition of annexin V was sufficient to block all rFVIII activity in the presence or absence of phospholipid vesicles, but could not block SI-Emi activity. Furthermore, addition of rivaroxaban, a direct FXa inhibitor, confirmed that detection of substrate cleavage was due to FXa activity. Conclusions: In the absence of phosphatidylserine-containing phospholipid vesicles, SI-Emi promoted the generation of FXa in plasma triggered with FXIa. The activity of BS-027125 was too low in this assay to clearly determine its phospholipid-independent activity. These results suggest SI-Emi has mis-regulated (PS-independent) procoagulant activity due to a lack of phospholipid localization of the antibody-FIXa-FX complex. Given the weak affinity of SI-Emi for its antigens, the exact mechanism enabling this activity is unclear. Further study of this phenomenon and its relevance to overall thrombin generation and in vivo activity are needed. Disclosures Aleman: Bioverativ, a Sanofi company: Employment. Jindal:Bioverativ, a Sanofi company: Employment. Leksa:Bioverativ a Sanofi company: Employment. Peters:Bioverativ a Sanofi company: Employment, Equity Ownership. Salas:Bioverativ a Sanofi company: Employment, Equity Ownership.

scholarly journals The Combination of Marzeptacog Alfa (Activated) or Eptacog Alfa (Activated) with Emicizumab Appears Comparable As Assessed By the Thrombin Generation Test in Hemophilia a Plasma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1112-1112
Author(s):  
Tom Knudsen ◽  
Brajesh Kumar ◽  
Frank Del Greco ◽  
Linda Neuman ◽  
Howard Levy ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Statistical Significance ◽  
Plasma Concentrations ◽  
Unmet Need ◽  
Surrogate Marker ◽  
Employment Equity ◽  
Thrombin Generation Assay ◽  
Equity Ownership

Background: Hemophilia patients treated prophylactically with emicizumab (Hemlibra®) may experience break-through bleeds or require additional hemostatic coverage for procedures or surgery. Currently available therapies including rFVIIa (Eptacog Alfa (Activated); NovoSeven®) and aPCC (FEIBA®) have been used with Hemlibra to treat bleeding or when additional coverage is required. While NovoSeven appears safe in combination with Hemlibra (HAVEN 1 to 4 clinical trials), thrombotic events have been observed with concurrent use of FEIBA and Hemlibra. While safe and efficacious when used as directed, NovoSeven must be infused intravenously. Ideally, for patients on subcutaneous (SC) prophylaxis with Hemlibra adjunct rFVIIa could be dosed SC too. Marzeptacog alfa (activated) (MarzAA) is a novel rFVIIa differentiated by increased potency and the ability to be administered SC to achieve pharmacologically relevant plasma concentrations. Thus, MarzAA provides a potential solution to address this unmet need in hemophilia therapy. Objective: Demonstrate the procoagulant potential of MarzAA, NovoSeven or FEIBA alone or in combination with Hemlibra using the thrombin generation assay in platelet poor hemophilia A (HA) plasma. The thrombin generation potential will therefore provide a surrogate marker to assess the potential safety and efficacy of SC MarzAA in combination with Hemlibra. Methods: A thrombin generation assay was performed using the PPP-Low Tissue Factor and phospholipid containing thrombin generation assay reagent (#TS31.00, Thrombinoscope, Stago). Citrated hemophilia A plasma was spiked with increasing concentrations (0, 25, 50, and 100 µg/mL) of Hemlibra together with various concentrations of each bypassing agent: MarzAA at 0, 0.1, 0.5, 1, 2.5, 5, and 10 µg/mL, NovoSeven at 1, 2.5, 5, 10, and 50 µg/mL or FEIBA at 0.25, and 0.50 IU/ml. Statistical significance was set at α = 0.05. Results: We assessed the relative potencies of MarzAA and NovoSeven in HA plasma. As expected, MarzAA demonstrated an approximate ten-fold increased potency vs NovoSeven. Both rFVIIa compounds increased peak thrombin generation in the HA plasma to the level of normal plasma and beyond. The effect of adding normalizing levels of MarzAA (1 µg/mL), NovoSeven (10 µg/mL) or FEIBA (0.5 IU/mL) to HA plasma containing clinically relevant concentrations of Hemlibra was evaluated (Fig 1). When correcting for the effect of Hemlibra alone, the increase in peak thrombin generation induced by FEIBA was significantly greater than that observed for both MarzAA and NovoSeven (P<0.002). In contrast, the observed increases in thrombin generation for MarzAA and NovoSeven in combination with Hemlibra were statistically indistinguishable. FEIBA was not tested at the highest clinically relevant concentration (2.0 IU/mL) as assay limitations were already approached at 0.5 IU/ml, corresponding to ~25% of the plasma concentration expected for a clinical FEIBA dose of 100 IU/kg. Furthermore, concentrations of MarzAA (5 µg/mL) or NovoSeven (50 µg/mL) 50-fold higher than expected after standard doses were required before peak thrombin generation became statistically indistinguishable from FEIBA at 0.5 IU/mL when all three compounds were evaluated in the presence of Hemlibra. Conclusion: As assessed by in vitro thrombin generation, equipotent concentrations of MarzAA and NovoSeven exhibit comparable characteristics when spiked into HA plasma containing Hemlibra at clinically relevant concentrations. Based on these data, MarzAA and NovoSeven are expected to behave similarly in combination with Hemlibra when dosed to achieve equipotent plasma concentrations. Figure 1 Disclosures Knudsen: Catalyst Biosciences: Employment, Equity Ownership. Kumar:Catalyst Biosciences: Employment, Equity Ownership. Del Greco:Catalyst Biosciences: Consultancy, Equity Ownership. Neuman:Catalyst Biosciences: Employment, Equity Ownership. Levy:Catalyst Biosciences: Employment, Equity Ownership. Blouse:Catalyst Biosciences: Employment, Equity Ownership.

scholarly journals A Novel and Potent Inhibitor of E-Selectin, GMI-1687, Attenuates Thrombus Formation and Augments Chemotherapeutic Intervention of AML in Preclinical Models Following Subcutaneous Administration

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4678-4678
Author(s):  
John Peterson ◽  
Myung-Gi Baek ◽  
Silvia Locatelli-Hoops ◽  
Ji-Won Lee ◽  
Lingquan Deng ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Tumor Model ◽  
Phase Iii ◽  
Therapeutic Activity ◽  
Preclinical Models ◽  
Employment Equity ◽  
Equity Ownership ◽  
High Bioavailability

Abstract Uproleselan (GMI-1271), an E-selectin antagonist, has been shown in preclinical models to disrupt activation of cell survival pathways in acute myeloid leukemia (AML), enhance chemotherapy efficacy, and improve survival. Uproleselan received FDA breakthrough therapy designation for adult relapsed/refractory AML in 2017 and Phase III studies are ongoing. In the present studies we report on the in vitro and in vivo comparative activities of an innovative high potency E-selectin antagonist, GMI-1687, a potential subcutaneously administered follow-on drug candidate to Uproleselan. The binding constant, association and dissociation rates of GMI-1687 to immobilized recombinant human (rh) E-selectin were determined by surface plasmon resonance (SPR) at 25oC. The KD of GMI-1687 was 2.4 nM, with Kon = 3 x 106 M-1s-1 and Koff = 1 x10-2 s-1. Under similar experimental conditions the KD of Uproleselan was 520 nM with Kon = 0.02 x 106 M-1s-1 and Koff = 1 x10-2 s-1. GMI-1687 was evaluated for its ability to inhibit binding of sialyl Lea to immobilized rh E-selectin. The median IC50 (n=6 independent assays) of GMI-1687 and Uproleselan in this assay was 15 and 550 nM, respectively. The in vitro activity of GMI-1687 to release adherent KG1a AML cells from E-selectin coated wells was also determined. GMI-1687 at 100 nM detached approximately 55% of adherent AML cells and was significantly different from Uproleselan at an identical concentration (38% detachment, P=0.0216). The percent bioavailability (%F) of GMI-1687 was evaluated in male Sprague-Dawley rats following intravenous (IV) and subcutaneous (SC) routes of administration at 5 mg/kg. The mean (+/- SD) SC %F for GMI-1687 was 126 +/- 3.8%. GMI-1687 also showed high bioavailability in CD-1 mice after SC administration of 0.58 mg/kg with %F = 132 +/-38. The in vivo therapeutic activity of GMI-1687 following SC administration was assessed in an acute model of inferior vena cava (IVC) thrombosis and a tumor model of AML.Immediately following the induction of a non-occlusive thrombosis via electrical stimulation (250 mAmp) of the IVC, cohorts of male C57BL/6J mice (n=5/group) were given a single SC injection of saline (0.1 mL); Uproleselan (40 mg/kg); or GMI-1687 (4 mg/kg, 0.4 mg/kg or 0.04 mg/kg), and twenty-four hrs post thrombus induction the IVC was harvested from all mice and thrombus weights were determined. Treatment with GMI-1687 decreased thrombus formation with significant inhibition at 0.04 mg/kg (92%, P<0.001 compared to saline control). The inhibition of thrombus formation with GMI-1687 dosed at 0.04 mg/kg was statistically indistinguishable from Uproleselan administered SC at 40 mg/kg (97% inhibition). The therapeutic activity of SC GMI-1687 was also observed in combination with chemotherapy in a U937 tumor model. Three days post IV injection of U937 tumor cells, bone marrow ablated, female NOD/SCID mice (n=10/group) were treated with saline (0.1 mL SC QDx14); GMI-1687 (0.04 mg/kg SC QDx14) alone; cytarabine (AraC 300 mg/kg IP QDx3) + daunorubicin (DNR 3 mg/kg IV QDx1), or the combination of GMI-1687 and AraC + DNR. All treatments were well tolerated. The median survival time (MST) of mice treated with AraC + DNR was 36 days and statistically different (P<0.001) to groups treated with saline (MST=22 days) or GMI-1687 alone (MST=23 days). Importantly, the therapeutic activity of AraC+DNR was significantly enhanced when combined with GMI-1687 (MST>47.5 days, P=0.0153 compared to AraC+DNR alone). In summary, a highly potent innovative antagonist of E-selectin, GMI-1687, has been produced that demonstrates high bioavailability following SC administration. SC injection of GMI-1687 shows significant activity in preclinical models previously reported for parenteral administration of Uproleselan, but at approximately 250-fold lower dose. GMI-1687 is therefore well-positioned for potential use in outpatient treatment settings where an E-selectin antagonist has therapeutic relevance. IND-enabling studies with GMI-1687 are currently underway. Disclosures Peterson: GlycoMimetics: Employment, Equity Ownership. Baek:GlycoMimetics: Employment, Equity Ownership. Locatelli-Hoops:GlycoMimetics: Employment, Equity Ownership. Lee:GlycoMimetics: Employment, Equity Ownership. Deng:GlycoMimetics: Employment, Equity Ownership. Stewart:GlycoMimetics: Employment, Equity Ownership. Smith:GlycoMimetics: Employment, Equity Ownership. Fogler:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Subcutaneously Administered RNAi Therapeutic (ALN-AT3) Targeting Antithrombin for Treatment of Hemophilia: Interim Phase 1 Study Results in Healthy Volunteers and Patients with Hemophilia a or B

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 693-693 ◽  
Author(s):  
Benny Sorensen ◽  
Tim Mant ◽  
Akin Akinc ◽  
Amy Simon ◽  
Lauren Melton ◽  
...  
Keyword(s):  
Factor Viii ◽  
Healthy Volunteers ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Thrombus Formation ◽  
Phase 1 ◽  
Study Drug ◽  
Relative Reduction ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction: Hemophilia A and B are congenital bleeding disorders caused by insufficient thrombin generation due to deficiency in factor VIII or IX, respectively. In the presence of normal levels of endogenous anticoagulants, deficiency of factor VIII or IX results in an imbalance of the hemostatic system toward a bleeding phenotype. ALN-AT3 is a subcutaneously administered investigational RNAi therapeutic aimed at reducing the levels of antithrombin (AT) with the purpose of increasing thrombin generation and thereby restoring hemostatic balance in hemophilia. Material and methods: Preclinical studies in hemophilia mouse models were used to investigate the ability of ALN-AT3 to reduce AT, increase thrombin generation, restore thrombus formation after microvascular laser injury, and control traumatic bleeding after saphenous vein transection. A study in an induced hemophilia A model in non-human primates was used to investigate the ability of ALN-AT3 to enhance thrombin generation in the setting of inhibitors to factor VIII. A Phase 1 clinical study in healthy volunteers and severe/moderate hemophilia A or B is ongoing. Part A, a single ascending dose study in healthy volunteers, has been completed and Part B, a multiple ascending dose study in hemophilia patients, is currently ongoing. Primary endpoints are safety and tolerability. Secondary endpoints are pharmacokinetics, relative reduction in levels of AT and change in thrombin generation as measured by Calibrated Automated Thrombin (CAT) generation. Results: ALN-AT3 treatment resulting in residual AT levels of 20-40% in hemophilia A and B mouse models increased thrombin generation, restored real-time localized thrombus formation in the laser-injury model comparable to treatment with full-length recombinant factor VIII. ALN-AT3 controlled traumatic bleeding in the saphenous vein model with an increase in number of hemostatic events equivalent to that achieved with infusion of 25 IU/kg full-length recombinant factor VIII. ALN-AT3 treatment targeting 20% residual AT levels normalized thrombin generation in non-human primates with induced high titer inhibitor hemophilia A. In Part A of the phase 1 study, a total of 4 human volunteer subjects were randomized to receive a single subcutaneous dose of ALN-AT3 0.03 mg/kg or placebo (3:1) and were followed for at least 70 days. No serious adverse events (SAEs) were observed. A total of 5 mild AEs were recorded. Four out of 5 mild AEs were considered unlikely/not related to study drug. One mild AE of temporary self-limiting headache was considered possibly related to study drug. In Part A, per protocol, the maximum allowable level of AT relative reduction was set at 40%. Results in healthy volunteers show that a single, low subcutaneous dose of ALN-AT3 at 0.03 mg/kg resulted in an up to 28-32% knockdown of AT activity at nadir, and the treatment effect was statistically significant relative to placebo (p < 0.01 by ANOVA). This led to a statistically significant (p < 0.01) increase in peak thrombin generation, that was temporally associated and consistent with the degree of AT knockdown. The AT reduction was stable and durable for up to 70 days post single dose. In the ongoing Part B, cohorts of 3 hemophilia patients will receive 3 weekly doses of ALN-AT3, with dosing commenced at 0.015 mg/kg. Up-to-date results from Part B will be presented. Conclusion: Collectively, these data suggest that the use of a novel RNAi therapeutic targeting AT is a promising approach for restoring hemostatic balance in hemophilia, and potentially, other bleeding disorders. Further, the subcutaneous route of administration, infrequent dosing, and applicability to persons with hemophilia who have inhibitors, make this a particularly encouraging potential therapy. Disclosures Sorensen: Alnylam Pharmaceuticals: Employment, Equity Ownership. Off Label Use: ALN-AT3 is an investigational drug for potential treatment of hemophilia. The data represent phase 1/2 data. Mant:Quintiles phase 1 unit, London: Employment. Akinc:Alnylam Pharmaceuticals: Employment. Simon:Alnylam Pharmaceuticals: Employment, Equity Ownership. Melton:Alnylam Pharmaceuticals: Employment, Equity Ownership. Lynam:Alnylam Pharmaceuticals: Employment. Strahs:Alnylam Pharmaceuticals: Employment, Equity Ownership. Sehgal:Alnylam Pharmaceuticals: Employment, Equity Ownership. Hutabarat:Alnylam Pharmaceuticals: Employment, Equity Ownership. Chaturvedi:Alnylam Pharmaceuticals: Employment, Equity Ownership. Barros:Alnylam Pharmaceuticals: Employment, Equity Ownership. Vaishnaw:Alnylam Pharmaceuticals: Employment, Equity Ownership. Investigators:Alnylam Pharmaceuticals: Clinical investigators in phase 1/2 study Other, Consultancy.

CTP-221, a Deuterated S-Enantiomer of Lenalidomide, Possesses Significantly Enhanced Immunomodulatory and Anti-Proliferative Properties Relative to the R-Enantiomer and to Racemic Lenalidomide.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2463-2463 ◽  
Author(s):  
Lijun Wu ◽  
Ara M. Aslanian ◽  
Julie F. Liu ◽  
Kristine Hogan ◽  
Roger Tung
Keyword(s):  
Multiple Myeloma ◽  
Clinical Efficacy ◽  
Whole Blood ◽  
Biological Activities ◽  
Employment Equity ◽  
Myeloma Cells ◽  
Tnf Α ◽  
Racemic Mixtures ◽  
Equity Ownership

Abstract Abstract 2463 Lenalidomide (Revlimid®) is an immunomodulatory drug (IMiD) currently approved for the treatment of 5q- myelodysplastic syndrome and multiple myeloma. The clinical efficacy of lenalidomide is thought to be related in part to enhanced T-cell co-stimulation and NK-cell activation via augmented IL-2 and IFN-γ production (Bartlett et al., 2004; Corral and Kaplan, 1999). Lenalidomide also inhibits TNF-α production in peripheral blood mononuclear cells (PBMCs) and whole blood, which may further contribute to its anti-tumor activity (Mueller et al., 1999). In addition to immunomodulatory effects, lenalidomide directly induces growth arrest and apoptosis in multiple myeloma cells, which is also recognized as a key mechanism of clinical efficacy (Mitsiades, 2002; Bartlett et al., 2004). IMiD-class compounds, including thalidomide, lenalidomide, and pomalidomide, have been developed as racemic mixtures of S- and R-enantiomers. The isolated enantiomers of thalidomide are known to have distinct biological activities. For example, the well-documented sedative effects of thalidomide are correlated with the R-enantiomer (Eriksson et al., 2000), whereas S-thalidomide exhibits enhanced potency for TNF-α inhibition and IL-2 induction compared to R-thalidomide (Mueller et al., 1999; Moreira et al., 2003; Macor, 2007). Due to facile in vivo conversion, isolated S- enantiomers of IMiDs have not been developed clinically. To our knowledge, it has not been previously reported whether lenalidomide has enantiospecific immunomodulatory, anti-proliferative, or toxicological properties. Given the therapeutic importance of lenalidomide, we explored a number of deuterium-substituted analogs of lenalidomide, either as racemic mixtures or as isolated S- and R-enantiomers, and studied them in several in vitro pharmacological assays. We found that in each case tested, deuterated racemic lenalidomide analogs were indistinguishable from non-deuterated lenalidomide across all the assays employed, including IL-2 induction in anti-CD3-stimulated PBMC, TNF-α inhibition in LPS-stimulated whole blood, and inhibition of proliferation of MM.1S human multiple myeloma cells. In contrast to deuterated racemic lenalidomide, CTP-221, an optimized deuterated S-lenalidomide analog, exhibited enhanced potency compared to racemic lenalidomide for IL-2 induction (2.7-fold), TNF-α inhibition (3.7-fold) and anti-proliferative (2.4-fold) activities in vitro. Interestingly, these enhancements in potency are greater than the maximal 2-fold enhancement one could expect from assessing an isolated active enantiomer in comparison to its racemate. These greater-than-expected enhancements in potency were consistently observed across all the assays comparing CTP-221 to lenalidomide, suggesting that deuterium substitution had additional effect(s) that drive increased potency. Furthermore, CTP-221 was significantly more potent than similarly deuterated R-lenalidomide in these assays (between 9.0 and 19.8-fold), demonstrating that the clinically relevant pharmacological activities of lenalidomide are primarily contained within the S-enantiomer. Finally, we found that CTP-221 was consistently more potent (1.2–2.0-fold) than non-deuterated S-lenalidomide. Taken together, these in vitro data demonstrate that deuterated racemic lenalidomide does not offer apparent advantages versus lenalidomide. However, the deuterated S-lenalidomide analog CTP-221 is significantly more potent than lenalidomide in key biological activities believed important for clinical efficacy. We plan to explore the toxicological properties of CTP-221 to assess its therapeutic window relative to lenalidomide. Disclosures: Wu: Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Aslanian:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Liu:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Hogan:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership. Tung:Concert Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Synergistic Effects of a Procoagulant Bispecific Antibody and Rescue Therapies on Thrombin Generation- a Potential Safety Risk

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4952-4952 ◽  
Author(s):  
Sabine Knappe ◽  
Rudolf Hartmann ◽  
Brahm Goldstein ◽  
Bruce M. Ewenstein ◽  
Leonard Valentino ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Synergistic Effects ◽  
Treatment Options ◽  
Phase Iii ◽  
Prospective Clinical Study ◽  
Normal Range ◽  
Experimental Conditions

Abstract Background: Investigational non-factor products, such as ACE 910 (Emicizumab), offer potential new treatment options for hemophilia patients with inhibitors. However, their uncertain and unregulated mechanisms of action raise multiple concerns around safety and efficacy in specific clinical contexts. As an antibody to FIXa and FX, ACE 910 lacks the inherent regulatory characteristics that are present in replacement factor and bypassing agents in the context of hemostasis. FEIBA is a plasma-derived, activated prothrombin complex concentrate developed to prevent and treat bleeding episodes in hemophilia A and B patients with inhibitors. There are more than 40 years of clinical experience with FEIBA. Extensive prospective clinical study data, as well as post-approval pharmacovigilance data demonstrated the product to be safe and highly effective. In an ongoing phase III study (NCT02622321), hemophilia A patients with inhibitors are treated with Emicizumab. To evaluate the treatment options for ACE910-treated patients experiencing breakthrough bleeding, we studied, in-vitro, the thrombin generation profile of various combinations of FEIBA with a biosimilar version of ACE910. Methods: A biosimilar antibody to Emicizumab (BS-Em) was expressed in mammalian cells, purified and extensively biochemically characterized. Therapeutic doses of BS-Em (200-600nM) in combination with several concentrations of FEIBA (0.1-1IU/ml) were analyzed in standard in-vitro thrombin generation (TG) experiments (1pM TF trigger) using 3 inhibitor patient plasma. A normal range of TG for the experimental conditions used was established using plasma from 34 healthy individuals. Results: The combination of FEIBA and BS-Em resulted in peak thrombin values of >500nM (600nM BS-Em/1IU/ml FEIBA) while the normal range was established to span peak thrombin levels of ~50-120nM thrombin. Titration experiments established that at 600nM BS-Em, FEIBA concentrations of >0.2IU/ml led to peak thrombin values 4- 10-fold higher than the normal range. Conclusions: Our in-vitro experiments demonstrate an excessive thrombin generation potential for the combination of BS-Em and FEIBA at concentrations expected to be generated in patients participating in this study. Caution and clinical judgement will be required when considering the use of ACE910 in hemophilia A patients with inhibitors as options to treat breakthrough bleeds if they occur might be reduced or lead to potential AE. Disclosures Knappe: Shire: Employment. Hartmann:Shire: Employment. Goldstein:Shire: Employment. Ewenstein:Shire: Employment. Valentino:Shire: Employment. Scheiflinger:Shire: Employment.

scholarly journals FEIBA® and Novoseven® Neutralize the Anticoagulant Effects of a Novel Small Molecule FXIa Inhibitor BMS-986177/JNJ-70033093 in Human Plasma and Whole Blood in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Matthew W Bunce ◽  
Zheng Huang Devine ◽  
Madhu Chintala
Keyword(s):  
Human Plasma ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Lag Time ◽  
Publicly Traded ◽  
Routine Prophylaxis ◽  
Control And Prevention ◽  
Bleeding Episodes

Background: FXIa inhibition is a promising antithrombotic drug target. BMS-986177/JNJ-70033093 (BMS-177/JNJ-3093) is a novel small molecular inhibitor of FXIa currently in Phase II clinical trials with the potential for reduced bleeding risk as compared to the currently approved oral anticoagulantsHowever, reversal of anticoagulation may still be required in patients who have uncontrolled or life-threatening bleeding or need an urgent surgical procedure. Aim: To evaluate the ability of nonspecific reversal agents (NSRAs) FEIBA®, NovoSeven®, Kcentra®, Profilnine®, BeneFix®, Novoeight®, and Cyklokapron® to neutralize the anticoagulation of BMS-177/JNJ-3093 in the activated partial thromboplastin time (aPTT), thromboelastography (TEG) and thrombin generation assay (TGA) in vitro using human plasma or whole blood. Method: aPTT and TEG were performed in human plasma and whole blood, respectively, using standard assay procedures. TGA was performed in human plasma using diluted kaolin aPTT reagent (1:10,000). JNJ-3093 was evaluated at different concentrations (0.3 -10 µM) to cover the anticipated exposures in the Phase II clinical trials. The NSRAs were evaluated at the anticipated concentrations according to the dosing information in their respective labels. Results: BMS-177/JNJ-3093 produced concentration dependent increases in aPTT (up to 4.4x at 10 μM); prolongations of lag time in TEG (2.6X); prolongations of lag time (3X) as well as reductions in peak thrombin generation (70%) in TGA. FEIBA® effectively neutralized the anticoagulant effects of JNJ-3093 in aPTT, TEG and TGA. NovoSeven® neutralized the BMS-177/JNJ-3093-induced prolongations in aPTT, prolongations in lag time in TEG and TGA assays and partially restored the peak thrombin generation in TGA. In contrast, all other NSRAs tested had negligible effects or did not show neutralization of anticoagulation induced by BMS-177/JNJ-3093 in the referenced assays Conclusion: These results demonstrate that FEIBA® and NovoSeven® can effectively neutralize the anticoagulant effects of BMS-177/JNJ-3093 in vitro. A clinical study is required to determine if these agents can reverse the anticoagulant effects of BMS-177/JNJ-3093 in patients. Table Disclosures Bunce: Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Huang Devine:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Chintala:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: FEIBA: hemophilia A and B patients with inhibitors for: control and prevention of bleeding episodes; use around the time of surgery; routine prophylaxis to prevent or reduce the frequency of bleeding episodes NovoSeven: Treatment of bleeding and prevention of bleeding for surgeries and procedures in adults and children with hemophilia A or B with inhibitors, congenital Factor VII (FVII) deficiency, and Glanzmanns thrombasthenia with a decreased or absent response to platelet transfusions; treatment of bleeding and prevention of bleeding for surgeries and procedures in adults with acquired hemophilia Kcentra: urgent reversal of acquired coagulation factor deficiency induced by vitamin K antagonist therapy in adult patients with need for urgent surgery/invasive procedure or acute major bleeding Profilnine: prevention and control of bleeding in patients with Factor IX deficiency due to hemophilia B BeneFix: control and prevention of bleeding episodes or peri-operative management in adult and pediatric patients with hemophilia B Novoeight: for use in adults and children with hemophilia A for control and prevention of bleeding, perioperative management, and routine prophylaxis to prevent or reduce the frequency of bleeding episodes Cyklokapron: patients with hemophilia for short-term use to reduce or prevent hemorrhage and reduce the need for replacement therapy during and following tooth extraction)

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