Tissue factor pathway inhibitor in activated prothrombin complex concentrates (aPCC) moderates the effectiveness of therapy in some severe hemophilia A patients with inhibitor

Abstract Abstract 3663 Bypassing agents such as activated prothrombin complex concentrates (APCC, FEIBA®) and recombinant activated factor VII (rFVIIa, NovoSeven®) are effective for most hemophiliac patients with inhibitors. While, some patients exhibit unresponsiveness to the treatment with APCC and/or rFVIIa, but their mechanisms remain unknown. We had a severe hemophilia A patient with inhibitor whose bleeding worsened despite of consecutive infusion of APCC. Switching from APCC to rFVIIa was very effective for his bleeding symptoms, and one-week cessation of bypassing agents had restored good response for APCC. Comprehensive coagulation assay such as thromboelastometry and thrombin generation test (TGT) provided us clear evidence of unresponsiveness to APCC. In particular, tissue factor (TF)-triggered TGT showed two significant features, the prolonged lag time and reduced peak thrombin level even after APCC infusion. Although FII, FVII(a), FIX, FX(a) and protein C contained in APCC were elevated in his plasmas after APCC infusion, increased amounts of these factors did not affect the parameters of TGT described above in vitro. We focused on a natural anticoagulant, tissue factor pathway inhibitor (TFPI), since the prolonged lag time in TF-triggered TGT might result from the impairment of FVII/TF-induced initial reaction of blood coagulation. In vitro experiment on the addition of TFPI to FVIII-deficient plasma with APCC showed similar inhibitory pattern in TGT. TFPI antigen levels (total and free forms) in his plasma actually increased above normal range after APCC infusion, whilst these levels unchanged after rFVIIa infusion and returned to the normal range after one-week cessation, speculating that TFPI might contributes to unresponsiveness to APCC. To confirm this, plasmas from several hemophiliac patients with APCC and/or rFVIIa infusion, including 4 patients with poor response pattern in TGT, were prepared. Among 12 pairs of plasmas (a pair; pre and post bypassing agents), each of 4 pairs were for APCC-poor response (APCC-PR), APCC-good response (APCC-GR), and rFVIIa-good response (FVIIa-GR). Free form TFPI antigen levels (normal; 15–35 ng/ml) increased after infusion in APCC-PR (pre/post; 38±4/51±3 ng/ml, p<0.05) and APCC-GR (28±4/37±4 ng/ml, p<0.05), but not increased in FVIIa-GR (23±3/21±3 ng/ml, p>0.05). Post-infusion levels in APCC-PR were significantly higher than those in APCC-GR (p<0.05). By adding anti-TFPI antibody, plasmas in APCC-PR showed marked increase of peak thrombin levels than those in APCC-GR in TGT, supporting that APCC-PR possessed more TFPI activity. Unexpectedly, ELISAs revealed that total TFPI were contained in APCC at 24±4 ng/unit (corresponded to 25≂f50% of physiological concentration), and 34% of them were free form, speculating that APCC infusion with ≂f90 units/kg appeared to increase free TFPI by ≂f15 ng/ml in plasma. Taken together, our results supported that TFPI contained in APCC attenuated the potentials of thrombin generation in hemophilia A patients with inhibitors, and some patients exhibited APCC-resistance due to TFPI accumulated by the consecutive use of APCC. Disclosures: Ogiwara: Baxter Hemophilia Scientific Research and Education Fund in Japan 2009: Research Funding. Nogami:Bayer Hemophilia Award Program 2009: Research Funding.

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Plasma Tissue Factor Pathway Inhibitor (TFPI) Levels in Healthy Subjects and Patients with Hemophilia A and B

Blood ◽

10.1182/blood.v126.23.4672.4672 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4672-4672 ◽

Cited By ~ 1

Author(s):

Jian-Ming Gu ◽

Chandra Patel ◽

Katalin Kauser

Keyword(s):

Tissue Factor ◽

Healthy Subjects ◽

Tissue Factor Pathway Inhibitor ◽

Hemophilia A ◽

Hemophilia B ◽

Severe Hemophilia ◽

Capture Assay ◽

Healthy Donors ◽

Tissue Factor Pathway

Abstract BAY 1093884 is a fully human monoclonal antibody against tissue factor pathway inhibitor (TFPI) developed as a potential bypass agent for patients with hemophilia with or without inhibitors. It restores insufficient thrombin burst, leading to stable clot formation in hemophilic conditions in vitro, and effectively stops bleeding in vivo. TFPI is a potent inhibitor of factor Xa (FXa) and the factor VIIa tissue factor complex in the extrinsic pathway. The majority of TFPI is associated with vascular endothelial cells. The mean plasma TFPI concentration in healthy individuals is ~70 ng/mL (1.6 nM) and about 80% of the circulating TFPI is bound to lipoproteins [Dahm, et al. Blood. 2003;101(11):4387-4392; Broze,et al. Front Biosci. 2012;17:262-280]. Some reports indicate that patients with hemophilia B have lower free TFPI levels than patients with hemophilia A, irrespective of phenotypic severity (Tardy-Poncet, et al. Haemophilia 2011;17:312-313). The objective of this study is to determine the plasma TFPI concentration in healthy donors and patients with hemophilia by a newly developed functional TFPI capture assay and to evaluate this assay with inhibition of TFPI by anti-TFPI neutralizing antibody (BAY 1093884) in vitro. A quantitative enzyme-linked immunosorbent assay using FXa as capture agent was developed and validated to measure TFPI levels in human plasma. The assay shows very good precision, accuracy, and reproducibility and should capture all coagulation-relevant forms of TFPI from plasma. Plasma TFPI was determined in 30 healthy donors (15 males and 15 females) and 30 patients with severe hemophilia (hemophilia A [n=12], hemophilia A with inhibitors [n=9], hemophilia B [n=9]). The plasma TFPI levels (mean ± SD) in healthy individuals, patients with severe hemophilia A without and with inhibitors, and severe hemophilia B were 59.5±18.4 ng/mL, 62.9±14.6 ng/mL, 47.3±4.3 ng/mL, and 68.1±8.8 ng/mL, respectively (Table 1). No statistical differences were found based on sex or race (Hispanic, African American, white) in the healthy population and between patients with hemophilia with and without inhibitors. TFPI levels were also not affected by addition of corn trypsin inhibitor (CTI) in citrate plasma. Furthermore, the concentration that inhibits 50% of TFPI levels (IC50) of anti-TFPI antibody (BAY 1093884) was determined to be 4.76 nM in normal human plasma using this assay. In conclusion,plasma TFPI does not appear to be affected by sex or race in healthy subjects, or the deficiency of factor VIII or IX in patients with hemophilia. The functional TFPI capture assay could potentially be used as a pharmacodynamic marker for monitoring plasma TFPI levels after the administration of anti-TFPI antibody and guide dosing strategies. Table 1. Plasma TFPI Levels in Healthy Subjects and Patients With Severe Hemophilia A and B HealthyHuman Donors(n=30) SevereHem A(n=12) Severe Hem AWith inhibitors(n=9) SevereHem B(n=9) TFPI, ng/mL Mean ± SD 59.5±18.4 62.9±14.6 47.3±4.3 68.1±8.8 Hem=hemophilia; TFPI=tissue factor pathway inhibitor. Disclosures Gu: Bayer HealthCare: Employment. Patel:Bayer HealthCare: Employment. Kauser:Bayer HealthCare LLC: Employment.

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Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

Blood ◽

10.1182/blood-2010-10-311936 ◽

2011 ◽

Vol 117 (20) ◽

pp. 5514-5522 ◽

Cited By ~ 87

Author(s):

Emily K. Waters ◽

Ryan M. Genga ◽

Michael C. Schwartz ◽

Jennifer A. Nelson ◽

Robert G. Schaub ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Hemophilia A ◽

Factor Viia ◽

Factor Xa ◽

Coagulation Factor ◽

Factor Ix ◽

Intrinsic Pathway ◽

Extrinsic Pathway ◽

Tissue Factor Pathway

Abstract Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

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A Neutralizing Monoclonal Antibody (PF-06741086) Against Tissue Factor Pathway Inhibitor in Combination with Activated Prothrombin Complex Concentrate Increases Hemostasis in Inhibitor Hemophilia Plasmas without Excessive Thrombin Generation

Blood ◽

10.1182/blood-2018-99-119831 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3779-3779

Author(s):

Swapnil Rakhe ◽

Sheryl Bowley ◽

John E. Murphy ◽

Debra D Pittman

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Prothrombin Complex Concentrate ◽

Hemophilia A ◽

Lag Time ◽

Hemophilia B ◽

Activated Prothrombin Complex Concentrate ◽

Tissue Factor Pathway

Abstract Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA). Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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Subcutaneous concizumab prophylaxis in hemophilia A and hemophilia A/B with inhibitors: phase 2 trial results

Blood ◽

10.1182/blood.2019001542 ◽

2019 ◽

Vol 134 (22) ◽

pp. 1973-1982 ◽

Cited By ~ 16

Author(s):

Amy D. Shapiro ◽

Pantep Angchaisuksiri ◽

Jan Astermark ◽

Gary Benson ◽

Giancarlo Castaman ◽

...

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Hemophilia A ◽

Factor X ◽

Phase 2 ◽

Phase 2 Trial ◽

Phase 2 Study ◽

Viii And Ix ◽

Tissue Factor Pathway

Tissue factor pathway inhibitor (TFPI) inhibits coagulation initiation, and TFPI inhibition enhances the activation of factor X by tissue factor. Shapiro et al report the results of a phase 2 study of prophylactic administration of the TFPI inhibitor concizumab for bleeding prevention in hemophilia, reporting efficacy in Factor VIII and IX deficiency even in patients with inhibitors to Factor VIII or IX.

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Efficacy of Anti-TFPI Antibody PF-06741086 Alone and in Combination with Activated Prothrombin Complex Concentrates in Mouse Hemophilia a Bleeding Model

Blood ◽

10.1182/blood-2018-99-118431 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2460-2460

Author(s):

Amey Barakat ◽

Reema Jasuja ◽

John E. Murphy ◽

Debra D Pittman

Keyword(s):

Blood Loss ◽

Mouse Model ◽

Tissue Factor ◽

Low Dose ◽

Hemophilia A ◽

Factor Xa ◽

Prothrombin Complex Concentrates ◽

Activated Prothrombin Complex Concentrates ◽

Dose Dependent ◽

Tail Clip

Abstract BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086. AIMS: Here, we directly compare the hemostatic effect of PF-06741086 alone, and in combination with aPCC in Hemophilia A mouse model using severe tail vein transection. METHODS: Male hemophilia A mice were dosed with a single intravenous dose of PF-06741086 (0.5, 1, 2 or 6 mg/kg) 30 minutes prior to a 3mm tail clip, or aPCC (50, 100 or 200 U/kg) was administered 5 minutes before the tail clip. Mice were also treated with a combined dose of 0.5 mg/kg anti-TFPI PF-06741086 and aPCC at 50, 100 or 200 U/kg. Blood was collected for 10 minutes and quantified against a standard curve of hemoglobin as volume blood loss. RESULTS: PF-06741086 demonstrated a dose dependent response in improving hemostasis in Hemophilia A mice after tail clip. PF-06741086 was able to restore hemostasis at 1 mg/kg (49%), and higher doses further improved hemostasis at 2 mg/kg (63%), and 6 mg/kg (78%). aPCC also demonstrated a dose dependent reduction in blood loss and improved hemostasis with all tested doses of 50 U/kg (25%), 100 U/kg (23%) and 200 U/kg (66%). At a dose of 0.5 mg/kg, PF-06741086 did not show any improvement in hemostasis over vehicle control. We used this dose for all combination studies with aPCC. Combined use of low dose PF-06741086 (0.5 mg/kg) and 100 U/kg aPCC shows a trend towards improvement in hemostasis compared to either drug alone. A higher dose of aPCC (200 U/kg) combined with low dose PF-06741086 (0.5 mg/kg) significantly reduces blood loss (86%) in Hemophilia A mice in tail clip model compared to saline, TFPI or aPCC alone used at the same dose of 0.5 mg/kg or 200 U/kg respectively. CONCLUSIONS: Prophylactic administration of PF-06741086 exhibits a dose response and improves hemostasis in an injury model in Hemophilia A mice. The addition of aPCC alone restores hemostasis at 200 U/kg and this effect was enhanced in combination with PF-06741086 in this mouse model. Disclosures Barakat: Pfizer: Employment. Jasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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Acute Efficacy of a Novel Anti-Tissue Factor Pathway Inhibitor Antibody BAY 1093884 in Hemophilia A Mouse Severe Tail Bleeding

Blood ◽

10.1182/blood.v126.23.3500.3500 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3500-3500 ◽

Cited By ~ 1

Author(s):

Yifan Xu ◽

Maria Koellenberger ◽

Volker Laux ◽

Katalin Kauser ◽

Derek Sim

Keyword(s):

Blood Loss ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Hemophilia A ◽

Human Monoclonal Antibody ◽

Factor Vii ◽

Tissue Factor Pathway ◽

Median Blood Loss

Abstract Tissue factor pathway inhibitor (TFPI) is the major inhibitor of the tissue factor initiated extrinsic coagulation pathway in blood and is intact in patients with hemophilia. The inhibition of TFPI may restore hemostasis in patients with hemophilia. BAY 1093884 is a fully human monoclonal antibody against TFPI developed as a bypass agent for hemophilia patients with or without inhibitors. It restores thrombin burst for stable clot formation in hemophilic conditions in vitro. The goal of these studies was to determine the in vivo acute efficacy of BAY 1093884 in the hemophilia A (HemA) mouse. In the first study, the acute efficacy of BAY 1093884 (3−100 mg/kg) was demonstrated and compared with full-length recombinant factor VIII (rFVIII; 10−100 IU/kg) by a HemA mouse tail clip model, in which blood loss from a severed tail tip was measured over 45 minutes after injury (n=12−27 mice/group). Naive C57/BL6 and HemA mice were used as positive and negative controls, respectively. Whereas isotype control antibody−treated HemA mice had median blood loss of 870 μL, increasing doses of BAY 1093884 to 50 and 100 mg/kg significantly reduced blood loss to a median of 55 and 5 μL. The dose required to reduce blood loss by 50% was 18 mg/kg, approximately equivalent to the efficacy of 20 IU/kg rFVIII. In a second study, we characterized the combined action of BAY 1093884 and activated recombinant factor VII (rFVIIa; n=10−25 mice/group). Low doses of BAY 1093884 (2.5 mg/kg) and rFVIIa (0.5 and 1.0 mg/kg) with minimal efficacies were tested. Untreated HemA mice had median blood loss of 860 μL. As stand-alone treatments, 2.5 mg/kg BAY 1093884, 0.5 mg/kg rFVIIa, and 1 mg/kg rFVIIa provided minimal blood loss protection, with bleeding volume reduced to 675, 830, and 770 μL, respectively. In comparison, the combination of 2.5 mg/kg BAY 1093884 with 0.5 mg/kg rFVIIa or 1.0 mg/kg rFVIIa reduced median blood loss to 215 and 35 μL, respectively. These results showed a combination effect of BAY 1093884 and rFVIIa in this severe acute efficacy model. These studies demonstrate that BAY 1093884 could potently reduce acute blood loss in HemA mice and may offer a new treatment option for hemophilia patients. Disclosures Xu: Bayer HealthCare LLC: Employment. Koellenberger:Bayer Pharma AG: Employment. Laux:Bayer Pharma AG: Employment. Kauser:Bayer HealthCare LLC: Employment. Sim:Bayer HealthCare LLC: Employment.

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