Factor XI promotes hemostasis in factor IX‐deficient mice

Essentials

• Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model.
• FIX‐deficient mice displayed a hemostatic defect and FXI‐deficient mice were similar to wild type mice.
• Infusion of FXI or over‐expression of FXI in FIX‐deficient mice improved hemostasis.
• FXI may affect the phenotype of FIX‐deficiency (hemophilia B).

Summary

Background

In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI‐deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI‐deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model.

Objectives

To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B).

Methods

Wild‐type mice and mice lacking either FIX (F9^?) or FXI (F11^?/?) were tested in the SVB model. The plasma levels of FXI in F11^?/? mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection.

Results

F9^? mice showed a significant defect in the SVB model, whereas F11^?/? mice and wild‐type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9^? mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX‐binding site also improved hemostasis in F9^? mice.

Conclusions

Although we were unable to demonstrate a hemostatic defect in F11^?/? mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9^? mice through FIX‐independent pathways.

     

Preclinical pharmacokinetics and biodistribution of subcutaneously administered glycoPEGylated recombinant factor VIII (N8‐GP) and development of a human pharmacokinetic prediction model

Essentials

• N8‐GP is an extended half‐life recombinant factor VIII (FVIII) for the treatment of hemophilia A.
• Subcutaneous (SC) FVIII dosing might reduce the treatment burden of prophylaxis.
• SC N8‐GP has a favorable PK profile in animal models and disappears from skin injection sites.
• Combined animal (SC) and clinical (IV) data suggest that daily SC dosing may provide prophylaxis.

Summary

Background

N8‐GP is an extended half‐life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous administration of FVIII may reduce the treatment burden of prophylaxis; however, standard FVIII products have low bioavailability after subcutaneous dosing in animals.

Objective

To evaluate the pharmacokinetics, effectiveness and local distribution of subcutaneously administered N8‐GP in preclinical models and predict the human pharmacokinetic (PK) profile.

Methods

The pharmacokinetics of subcutaneously administered N8‐GP were evaluated in FVIII knockout (F8‐KO) mice and cynomolgus monkeys; a human PK prediction model in hemophilia A patients was developed. The hemostatic effect was evaluated in a tail vein bleeding model in F8‐KO mice. The injection‐site distribution and absorption of subcutaneously administered N8‐GP were assessed in F8‐KO mice by the use of temporal fluorescence imaging and immunohistochemistry.

Results

Subcutaneously administered N8‐GP had a bioavailability, a first‐order absorption rate and a half‐life, respectively, of 24%, 0.094 h^?1 and 14 h in F8‐KO mice, and 26%, 0.33 h^?1 and 15 h in cynomolgus monkeys. A dose‐dependent effect of subcutaneously administered N8‐GP on blood loss was observed in mice. A minimal amount of N8‐GP was detected at the injection site 48–72 h after single or multiple dose(s) in F8‐KO mice. Subcutaneously administered N8‐GP was localized to the skin around the injection site, with time‐dependent disappearance from the depot. PK modeling predicted that subcutaneously administered N8‐GP at a daily dose of 12.5 IU kg^?1 will provide FVIII trough levels of 2.5–10% in 95% of patients with severe hemophilia A.

Conclusions

Subcutaneously administered N8‐GP may provide effective hemophilia A prophylaxis. A phase I clinical trial is underway to investigate this possibility.

     

Anti‐C1 domain antibodies that accelerate factor VIII clearance contribute to antibody pathogenicity in a murine hemophilia A model

Essentials

• Inhibitor formation remains a challenging complication of hemophilia A care.
• The Bethesda assay is the primary method used for determining bleeding risk and management.
• Antibodies that block factor VIII binding to von Willebrand factor can increase FVIII clearance.
• Antibodies that increase clearance contribute to antibody pathogenicity.

Summary

Background

The development of neutralizing anti‐factor VIII (FVIII) antibodies remains a challenging complication of modern hemophilia A care. In vitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms.

Objectives

To evaluate FVIII clearance in vivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype.

Methods

FVIII^?/? or FVIII^?/?/von Willebrand factor (VWF)^?/? mice were infused with anti‐FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured.

Results

Pathogenic anti‐C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII–mAb complexes in FVIII^?/? mice but not in FVIII^?/?/VWF^?/? mice. Additionally, pathogenic anti‐C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII^?/? mice. Anti‐C1, anti‐C2 and anti‐A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti‐C1 mAbs partially corrected blood loss in FVIII^?/? mice.

Conclusions

A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII–mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophilia A and low‐titer inhibitors.

     

Factor VIII products and inhibitor development in previously treated patients with severe or moderately severe hemophilia A: a systematic review

Essentials

• Data on product‐related immunogenicity in previously treated haemophilia A patients is scarce.
• A systematic review and meta‐analysis of all currently available evidence was conducted.
• The overall incidence rate was 2.06 per 1000 person‐years (95% confidence interval: 1.06‐4.01).
• Some recombinant factor VIII products were associated with increased immunogenicity.

Summary

Background

Patients with severe hemophilia A who have been treated extensively with factor VIII products have a low but potentially serious risk of inhibitor development. It is unknown why these patients develop inhibitors, and data on product‐related immunogenicity are scarce.

Aims

To summarize the currently available evidence on the relationship between inhibitor development and recombinant FVIII product type in previously treated patients (PTPs) with severe hemophilia A.

Methods

Longitudinal studies were included that reported on de novo inhibitor formation in patients with baseline FVIII activity levels of < 0.02 IU mL^?1 who had been treated with FVIII for at least 50 days. Pooled incidence rates of inhibitor development according to product types were calculated with a random intercept Poisson regression model.

Results

Forty‐one independent cohorts were included; 39 patients developed de novo inhibitors during 19 157 person‐years of observation. The overall incidence rate was 2.06 per 1000 person‐years, with a 95% confidence interval (CI) of 1.06–4.01. According to product type, the pooled incidence rates were 0.99 (95% CI 0.37–2.70) per 1000 person‐years for patients treated with Advate, 5.86 (95% CI 0.25–134.92) per 1000 person‐years for those treated with Kogenate/Helixate, 1.35 (95% CI 0.66–2.77) per 1000 person‐years for those treated with Kogenate FS/Helixate NexGen, 12.05 (95% CI 1.53–94.78) per 1000 person‐years for those treated with Refacto, and 4.64 (95% CI 0.82–26.43) per 1000 person‐years for those treated with Refacto AF.

Conclusion

These results suggest that some products may be associated with increased immunogenicity. However, the low incidence of inhibitors in PTPs and the differences in study design may cause significant variation in estimates of risk.

     

Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti‐factor IXa/factor X bispecific antibody emicizumab

Essentials

• The activated partial prothrombin time (aPTT) cannot predict the activity of emicizumab (Emi).
• Adjusted clot waveform analyses using a prothrombin time (PT)/aPTT initiator were developed.
• Activity of Emi in the co‐presence of factor VIII or bypassing agents was quantified.
• This assay is useful for assessing coagulation potential in Emi‐treated hemophilia A.

Summary

Background

Emicizumab is an anti‐activated factor IX/FX bispecific antibody that mimics activated FVIII cofactor function. Emicizumab does not require activation by thrombin, and its effect on shortening the activated partial thromboplastin time (APTT) is much greater than that of FVIII. Therefore, the APTT has limited utility in hemophilia A (HA) patients treated with emicizumab.

Aim

To evaluate the global coagulation potential of emicizumab.

Methods

Clot waveform analysis (CWA) with prothrombin time (PT)/APTT mixed reagents was used to define hemostatic monitoring protocols in HA patients. A modified parameter, adjusted‐|min1| (Ad|min1|), was developed. Maximum and minimum percentage transmittance were defined as 100% and 0% in the precoagulation and postcoagulation phases, respectively. Ad|min1| was calculated as an index of the maximum velocity of the coagulation process.

Results

Ad|min1| obtained with mixed‐trigger reagent (PT/APTT/buffer, 1 : 15 : 135) in the presence of emicizumab optimally corresponded to the conversion rate estimated in animals; 0.2–0.4 IU dL^?1 equivalent FVIII per 1 μg mL^?1 emicizumab). Ex vivo addition of emicizumab to HA plasma with or without inhibitors resulted in concentration‐dependent increases in Ad|min1|, with some individual variations. The addition of various concentrations of FVIII to HA plasma mixed with emicizumab resulted in dose‐dependent increases in Ad|min1|. Similarly, mixtures of activated prothrombin complex concentrate and emicizumab added to HA plasma resulted in dose‐dependent increases in Ad|min1|. In contrast, enhanced coagulation potential appeared to be better defined by the clot time than by Ad|min1| in experiments using recombinant activated FVII.

Conclusion

The PT/APTT reagent‐triggered adjusted CWA could provide a useful means of assessing global coagulation potential in emicizumab‐treated HA patients, with enhanced activity neither masking nor being masked by FVIII or bypassing agents.

     

Auto‐ and alloantibodies against factor XIII: laboratory diagnosis and clinical consequences

Summary

Acquired FXIII deficiencies caused by autoantibodies against FXIII subunits represent rare but very severe bleeding diatheses. Alloantibodies in FXIII‐deficient patients also cause life‐threatening bleeding complications, but they develop extremely rarely. In this review we provide an overview of the diagnosis and classification of anti‐FXIII antibodies and analyze 48 patients with autoimmune FXIII deficiency and four additional FXIII‐deficient patients who developed anti‐FXIII alloantibody. The patients were collected from peer‐reviewed publications from which relevant data could be extracted. With the exception of two cases the antibodies were directed against FXIII‐A. The difficulties in the diagnosis of FXIII deficiency in the presence of anti‐FXIII antibodies are discussed and a scheme for the functional classification of the anti‐FXIII antibodies is recommended. The three main categories are neutralizing and non‐neutralizing antibodies and antibodies with combined effect. The methods being used for detecting and quantifying the inhibitory effect on FXIII activation and on the transglutaminase activity of activated FXIII are summarized and techniques for the classification of neutralizing anti‐FXIII antibodies are outlined. The importance of clearance studies in these cases is emphasized. Binding assays, useful for the identification of non‐neutralizing and combined type antibodies, were collected from the literature and their informative power is demonstrated by examples. The most frequently occurring bleeding symptoms in patients with anti‐FXIII antibodies were soft tissue bleeding; intracranial bleedings also occurred, but less frequently than in inherited FXIII deficiency. Treatment of such patients is extremely challenging; the main aim should be eradication of the antibody.

     

The perils of inhibiting deficient factors

Essentials

• Anticoagulation in patients with factor X deficiency is an evidence‐poor area.
• A patient with factor X deficiency was anticoagulated with warfarin followed by rivaroxaban.
• Warfarin may be a safer anticoagulant option than rivaroxaban in hereditary factor X deficiency.
• A baseline coagulation screen should be performed prior to commencement of anticoagulation.

Summary

We report a case of a previously undiagnosed factor X deficiency in an 83‐year‐old man who had no previous bleeding history despite multiple hemostatic challenges. He was anticoagulated with warfarin for atrial fibrillation without bleeding complications; however, major hemorrhage occurred soon after a switch to rivaroxaban.

     

Polymorphism in dural arteriovenous fistula: matrix metalloproteinase‐2‐1306 C/T as a potential risk factor for sinus thrombosis

Essentials

• Sinus thrombosis may play a crucial role in development of dural arteriovenous fistula (DAVF).
• Little is known about the association between gene polymorphism and the development of DAVF.
• MMP‐2‐1306 C/T showed a higher prevalence rate in DAVF cases with sinus thrombosis.
• MMP‐2‐1306C/T polymorphism is likely a potential risk factor for sinus thrombosis in DAVF.

Summary

Background

Dural arteriovenous fistula (DAVF) is a rare but important cerebrovascular disorder in adults. Little is known about the molecular genetic pathogenesis underlying DAVF development.

Objectives

To investigate the associations of gene polymorphisms and DAVF.

Materials and Methods

By the use of real‐time PCR genotyping, seven single‐nucleotide polymorphisms (SNPs) of angiogenesis‐related genes were analyzed in 72 DAVF patients. Pertinent clinical and imaging data were subgrouped on the basis of location (cavernous sinus versus lateral sinus), lesions (single versus multiple), cerebral venous reflux (CVR) grading (Borden I versus Borden II/III), and sinus thrombosis (with versus without).

Results

We found that individuals carrying the polymorphic allele of matrix metalloproteinase (MMP)‐2‐1306 C/T (rs243865) had a significantly increased risk of sinus thrombosis in DAVF (odds ratio 6.2; 95% confidence interval 1.7–22.9). There was a weak difference in associations of tissue inhibitor of metalloproteinase (TIMP)‐2 (rs2277698) gene polymorphism and DAVF patients subgrouped by CVR grading.

Conclusions

These preliminary results indicate that MMP‐2‐1306 C/T, but not MMP‐9, TIMP‐1, TIMP‐2, and vascular endothelial growth factor A SNP variants, is a risk factor for the development of sinus thrombosis in DAVF patients.

     

Mild antithrombin deficiency and risk of recurrent venous thromboembolism: results from the MEGA follow‐up study

Essentials

• Mild antithrombin deficiency may increase the risk of recurrent venous thromboembolism (VTE).
• In a cohort study, we stratified patients with VTE to various cut‐off antithrombin levels.
• A 1.6–3.7‐fold increased risk of recurrent VTE was observed in the lowest antithrombin categories.
• Mild antithrombin deficiency (activity < 5th percentile of normal) increases recurrent VTE risk.

Summary

Background

Mild antithrombin deficiency (previously defined as antithrombin activity below 70% or 80%) has been associated with a 2.4–3.5‐fold increased risk of recurrent venous thromboembolism (VTE). This finding may have implications for duration of antithrombotic therapy in VTE patients with mild antithrombin deficiency.

Objectives

To externally validate whether mild antithrombin deficiency is a risk factor for recurrent VTE.

Methods

In a population‐based cohort study, patients with a first VTE (n = 2357) were stratified according to percentile cut‐off antithrombin levels (< 5th [< 87%], 5–10th [87–92%], > 10th percentile [> 92%]) and functional antithrombin levels (< 70%, 70–80%, > 80%).

Results

During a median follow‐up of 7.4 years, 361 recurrent events occurred (incidence rate, 2.5/100 patient‐years). We observed an increased risk of recurrent VTE in the lowest antithrombin activity category (< 5th percentile; < 87%) as compared with antithrombin activity that was > 10th percentile (> 92%), with an adjusted hazard ratio (HR) of 1.5 (95%CI, 1.0–2.3). When analyses were stratified to antithrombin cut‐off criteria of< 70% vs. patients with antithrombin activity > 80%, the adjusted HR for venous recurrence was 3.7 (95% CI, 1.4–9.9). Mild antithrombin deficiency was able to predict recurrent VTE over at least 8 years of follow‐up and the association remained present when the population was stratified to the presence or absence of thrombosis risk factors. Restriction analyses, where patients who used anticoagulation at time of blood draw and those who reported drinking ≥ 5 glasses alcohol daily were excluded, did not materially affect these outcomes.

Conclusion

This study confirms that mild antithrombin deficiency is a risk factor for recurrent VTE.

     

Efficacy,safety and pharmacokinetics of a new high‐purity factor X concentrate in women and girls with hereditary factor X deficiency

Essentials

• Plasma‐derived factor X concentrate (pdFX) is used to treat hereditary factor X deficiency.
• pdFX pharmacokinetics, safety and efficacy were assessed in factor X‐deficient women/girls.
• Treatment success rate was 98%; only 6 adverse events in 2 subjects were possibly pdFX related.
• On‐demand pdFX 25 IU kg^?1 was effective and safe in women/girls with factor X deficiency.

Summary

Background

A high‐purity, plasma‐derived factor X concentrate (pdFX) has been approved for the treatment of hereditary FX deficiency, an autosomal recessive disorder.

Objective

To perform post hoc assessments of pdFX pharmacokinetics, safety and efficacy in women and girls with hereditary FX deficiency.

Patients/Methods

Subjects aged ≥ 12 years with moderate/severe FX deficiency (plasma FX activity of < 5 IU dL^?1) received on‐demand or preventive pdFX (25 IU kg^?1) for ≤ 2 years.

Results

Of 16 enrolled subjects, 10 women and girls (aged 14–58 years [median, 25.5 years]) received 267 pdFX infusions. Mean monthly infusions per subject were higher among women and girls (2.48) than among men and boys (1.62). In women and girls, 132 assessable bleeding episodes (61 heavy menstrual bleeds, 47 joint bleeds, 15 muscle bleeds, and nine other bleeds) were treated with pdFX, with a 98% treatment success rate versus 100% in men and boys. Mean pdFX incremental recovery was similar in the two groups (2.05 IU dL^?1 versus 1.91 IU dL^?1 per IU kg^?1), as was the mean half‐life (29.3 h versus 29.5 h). Of 142 adverse events in women and girls, headache was the most common (12 events in six subjects). Six events (two infusion‐site erythema, two fatigue, one back pain, one infusion‐site pain) in two subjects were considered to be possibly pdFX‐related. Following the trial, pdFX was used to successfully maintain hemostasis in two subjects undergoing obstetric delivery.

Conclusions

pdFX was well tolerated and effective in women and girls with FX deficiency. Although women and girls had different bleeding symptoms and sites than men and boys, their pdFX pharmacokinetic profile was comparable.

     

Functional characterization of recombinant snake venom rhodocytin: rhodocytin mutant blocks CLEC‐2/podoplanin‐dependent platelet aggregation and lung metastasis

Essentials

• We generated recombinant rhodocytin that could aggregate platelets via CLEC‐2.
• Recombinant wild‐type rhodocytin formed heterooctamer with four α‐ and β‐subunits.
• Asp 4 in α‐subunit of rhodocytin was required for binding to CLEC‐2.
• Inhibitory mutant of rhodocytin blocked podoplanin‐dependent hematogenous metastasis.

Summary

Background

Rhodocytin, a disulfide‐linked heterodimeric C‐type lectin from Calloselasma rhodostoma consisting of α‐subunits and β‐subunits, induces platelet aggregation through C‐type lectin‐like receptor 2 (CLEC‐2). CLEC‐2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell‐induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti‐CLEC‐2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation.

Objective

To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti‐CLEC‐2 drug based on the findings.

Methods

We used Chinese hamster ovary cells to express recombinant rhodocytin (wild‐type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC‐2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN‐induced metastasis in an experimental lung metastasis mouse model.

Results

Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α‐subunits of rhodocytin was required for CLEC‐2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC‐2 without inducing platelet aggregation, and blocked CLEC‐2–PDPN interaction‐dependent platelet aggregation and experimental lung metastasis.

Conclusion

These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC‐2.

     

Routine measurements of factor VIII activity and inhibitor titer in the presence of emicizumab utilizing anti‐idiotype monoclonal antibodies

Essentials

• Emicizumab (Emi) affects the APTT‐based assays of factor (F)VIII activity and inhibitor titer.
• A mixture of two anti‐Emi monoclonal antibodies (mAb) effectively neutralized the Emi activity.
• Anti‐Emi mAbs completely eliminated the influence of Emi on FVIII activity and inhibitor titer.
• The inclusion of anti‐Emi mAbs in routine FVIII assays would be useful for Emi‐treated patients.

Summary

Background

Emicizumab is an anti‐factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT‐based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab.

Aim

To establish a reliable APTT‐based assay to measure FVIII in the presence of emicizumab.

Methods

Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one‐stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti‐idiotype mAbs to emicizumab (anti‐emicizumab mAbs) were prepared, rcAQ8 to anti‐FIXa‐Fab and rcAJ540 to anti‐FX‐Fab.

Results

The combined anti‐idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 μm = ~150 μg mL^?1) at all reference levels of FVIII. The addition of anti‐emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti‐FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti‐FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti‐idiotype mAbs, allowing accurate assessment of inhibitor titers.

Conclusion

The in vitro inclusion of anti‐emicizumab mAbs in the standard one‐stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers.

     

A first‐in‐human study of the safety,tolerability, pharmacokinetics and pharmacodynamics of PF‐06741086, an anti‐tissue factor pathway inhibitor mAb,in healthy volunteers

Essentials

• Tissue factor pathway inhibitor (TFPI) is an antagonist of FXa and the TF‐FVIIa complex.
• PF‐06741086 is an IgG₁ monoclonal antibody that targets the Kunitz‐2 domain of TFPI.
• Single doses of PF‐06741086 were evaluated in a phase 1 study in healthy volunteers.
• Data from this study support further investigation of PF‐06741086 in individuals with hemophilia.

Summary

Background

Tissue factor pathway inhibitor (TFPI) is a protease inhibitor of the tissue factor–activated factor VII complex and activated FX. PF‐06741086 is a mAb that targets TFPI to increase clotting activity.

Objectives

This study was a randomized, double‐blind, sponsor‐open, placebo‐controlled, single intravenous or subcutaneous dose escalation study to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of PF‐06741086.

Patients/Methods

Volunteers who provided written informed consent were assigned to cohorts with escalating dose levels. Safety endpoints included treatment‐emergent adverse events (TEAEs), infusion/injection site reactions, vital signs, electrocardiogram, and coagulation and hematology laboratory parameters. Pharmacokinetic (PK) and pharmacodynamic (PD) endpoints included exposures of PF‐06741086 in plasma and measures of PF‐06741086 pharmacology, respectively.

Results

Forty‐one male volunteers were recruited overall. Thirty‐two were dosed with PF‐06741086 from 30 mg subcutaneously to 440 mg intravenously. All doses were safe and well tolerated. TEAEs were mild or moderate in severity, laboratory abnormalities were transient, there were no serious adverse events, there were no infusion/injection site reactions, and no dose escalation stopping criteria were met. Plasma exposures of PF‐06741086 increased greater than proportionally with dose under the same dosing route. Coagulation pharmacology was demonstrated via total TFPI, dilute prothrombin time, D‐dimer, prothrombin fragment 1 + 2 and thrombin generation assay parameters.

Conclusions

Single doses of PF‐06741086 at multiple dose levels were safe and well tolerated in a healthy adult male population. The safety, PK and PD data from this study support progression to a multiple‐dose study in hemophilic patients.

     

Carboxypeptidase U (CPU,carboxypeptidase B2, activated thrombin‐activatable fibrinolysis inhibitor) inhibition stimulates the fibrinolytic rate in different in vitro models

Essentials

• AZD9684 is a potent inhibitor of carboxypeptidase U (CPU, TAFIa, CPB2).
• The effect of AZD9684 on fibrinolysis was investigated in four in vitro systems.
• The CPU system also attenuates fibrinolysis in more advanced hemostatic systems.
• The size of the observed effect on fibrinolysis is dependent on the exact experimental conditions.

Summary

Background

Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin‐activatable fibrinolysis inhibitor) is a basic carboxypeptidase that attenuates fibrinolysis. This characteristic has raised interest in the scientific community and pharmaceutical industry for the development of inhibitors as profibrinolytic agents.

Objectives

Little is known about the contribution of CPU to clot resistance in more advanced hemostatic models, which include blood cells and shear stress. The aim of this study was to evaluate the effects of the CPU system in in vitro systems for fibrinolysis with different grades of complexity.

Methods

The contribution of the CPU system was evaluated in the following systems: (i) plasma clot lysis; (ii) rotational thromboelastometry (ROTEM) in whole blood; (iii) front lysis with confocal microscopy in platelet‐free and platelet‐rich plasma; and (iv) a microfluidic system with whole blood under arterial shear stress. Experiments were carried out in the presence or absence of AZD9684, a specific CPU inhibitor.

Results

During plasma clot lysis, addition of AZD9684 resulted in 33% faster lysis. In ROTEM, the lysis onset time was decreased by 38%. For both clot lysis and ROTEM, an AZD9684 dose‐dependent response was observed. CPU inhibition in front lysis experiments resulted in 47% and 50% faster lysis for platelet‐free plasma and platelet‐rich plasma, respectively. Finally, a tendency for faster lysis was observed only in the microfluidic system when AZD9684 was added.

Conclusions

Overall, these experiments provide novel evidence that the CPU system can also modulate fibrinolysis in more advanced hemostatic systems. The extent of the effects appears to be dependent upon the exact experimental conditions.

     

High and long‐term von Willebrand factor expression after Sleeping Beauty transposon‐mediated gene therapy in a mouse model of severe von Willebrand disease

Essentials

• von Willebrand disease (VWD) is the most common inherited bleeding disorder.
• Gene therapy for VWD offers long‐term therapy for VWD patients.
• Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice.
• Liver‐directed transposons support sustained VWF expression with suboptimal multimerization.

Summary

Background

Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short‐term solution. Gene therapy offers the potential for a long‐term treatment for VWD.

Objectives

To develop an integrative Sleeping Beauty (SB) transposon‐mediated VWF gene transfer approach in a preclinical mouse model of severe VWD.

Methods

We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf^?/? mice by combining a liver‐specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery.

Results

The sandwich SB transposon was suitable to deliver the full‐length mVWF cDNA (8.4 kb) and supported supra‐physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride‐induced liver regeneration. Analysis of integration sites by high‐throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long‐term expression of supra‐physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long‐term expression of VWF by hepatocytes resulted in relatively reduced amounts of high‐molecular‐weight multimers, potentially limiting its hemostatic efficacy.

Conclusions

Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type‐specific targeting is yet to be achieved.

     

Abrogating fibrinolysis does not improve bleeding or rFVIIa/rFVIII treatment in a non‐mucosal venous injury model in haemophilic rodents

Essentials

• The efficacy of systemic antifibrinolytics for hemophilic non‐mucosal bleeding is undetermined.
• The effect of systemically inhibiting fibrinolysis in hemophilic mice and rats was explored.
• Neither bleeding nor the response to factor treatment was improved after inhibiting fibrinolysis.
• The non‐mucosal bleeding phenotype in hemophilia A appears largely unaffected by fibrinolysis.

Summary

Background

Fibrinolysis may exacerbate bleeding in patients with hemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain hemostatic control. Although antifibrinolytic drugs have been proven to be effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non‐mucosal tissues remain an open question of significant clinical interest.

Objective

To determine whether inhibiting fibrinolysis improves the outcome in non‐mucosal hemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard ex vivo clotting/fibrinolysis assay can be used as a predictive surrogate for in vivo efficacy.

Methods

A highly sensitive TVT model was employed in hemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in non‐mucosal tissue. In mice, induced and congenital hemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid [TXA]). In hemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry evaluation of the same animals to gauge the predictive value of such assays.

Results

The beneficial effect of systemic TXA therapy observed ex vivo could not be confirmed in vivo in hemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes encoding plasminogen or tissue‐type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in hemophilic mice.

Conclusions

The findings here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of HA rodents in non‐mucosal tissues.

     

Platelet rescue by macrophage depletion in obese ADAMTS‐13‐deficient mice at risk of thrombotic thrombocytopenic purpura

Essentials

• Obesity is a potential risk factor for development of thrombotic thrombocytopenic purpura (TTP).
• Obese ADAMTS‐13‐deficient mice were triggered with von Willebrand factor (VWF).
• Depletion of hepatic and splenic macrophages protects against thrombocytopenia in this model.
• VWF enhances phagocytosis of platelets by macrophages, dose‐dependently.

Summary

Background

Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS‐13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets.

Objectives

To evaluate the role of obesity and ADAMTS‐13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia.

Methods

Lean or obese ADAMTS‐13‐deficient (Adamts‐13^?/?) and wild‐type (WT) mice were injected with 250 U kg^?1 of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts‐13^?/? mice by injection of clodronate‐liposomes, 48 h before injection of rVWF.

Results

Obese Adamts‐13^?/? mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts‐13^?/? mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts‐13^?/? mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts‐13^?/? mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose‐dependent increase of platelet phagocytosis.

Conclusions

Obese Adamts‐13^?/? mice are more susceptible to the induction of TTP‐related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

     

Phase 1, single‐dose escalating study of marzeptacog alfa (activated), a recombinant factor VIIa variant,in patients with severe hemophilia

Essentials

• Marzeptacog alfa (activated) [MarzAA] is a novel variant of activated human factor VII.
• A phase 1 dose escalation trial of MarzAA was conducted in subjects with severe hemophilia.
• MarzAA was safe and tolerated at intravenous doses up to 30 μg kg^?1
• Data observed support further trials for hemophilia patients with inhibitors to factors VIII/IX.

Summary

Background

Marzeptacog alfa (activated) (MarzAA), a new recombinant activated human factor VII (rFVIIa) variant with four amino acid substitutions, was developed to provide increased procoagulant activity and a longer duration of action in people with hemophilia.

Objectives

To investigate the safety, tolerability, immunogenicity, pharmacokinetics (PK) and pharmacodynamics (PD) of single ascending intravenous bolus doses of MarzAA in non‐bleeding patients with congenital hemophilia A or B with or without inhibitors.

Methods

This international, phase 1, open‐label study (NCT01439971) enrolled males aged 18–64 years with severe hemophilia A or B, with or without FVIII or FIX inhibitors. Subjects were assigned to single‐dose MarzAA cohorts (0.5, 4.5, 9, 18 or 30 μg kg^?1). Blood sampling was performed predose and postdose, and subjects were monitored for 60 days postdose. Safety endpoints included adverse events, vital sign changes, electrocardiograms, laboratory abnormalities, and immunogenicity; secondary endpoints included evaluation of PK and PD.

Results

Overall, in 25 patients, MarzAA was well tolerated at all dose levels tested, and was not associated with dose‐limiting toxicity. No treatment‐emergent severe or serious adverse events occurred. MarzAA showed linear dose–response PK across the 4.5–30 μg kg^?1 dose range, with a terminal half‐life of ? 3.5 h. Dose‐dependent shortening of the activated partial thromboplastin time and prothrombin time, and evidence of an increase in peak thrombin as determined with a thrombin generation assay, were observed at all doses.

Conclusions

MarzAA was tolerated at doses up to 30 μg kg^?1. The safety profile and pharmacological effects observed support further clinical trials for the treatment of hemophilic patients with inhibitors.

     

Correction of a dominant‐negative von Willebrand factor multimerization defect by small interfering RNA‐mediated allele‐specific inhibition of mutant von Willebrand factor

Essentials

• Substitution therapy for von Willebrand (VW) disease leaves mutant VW factor (VWF) unhindered.
• Presence of mutant VWF may negatively affect phenotypes despite treatment.
• Inhibition of VWF by allele‐specific siRNAs targeting single‐nucleotide polymorphisms is effective.
• Allele‐specific inhibition of VWF p.Cys2773Ser improves multimerization.

Summary

Background

Treatment of the bleeding disorder von Willebrand disease (VWD) focuses on increasing von Willebrand factor (VWF) levels by administration of desmopressin or VWF‐containing concentrates. Both therapies leave the production of mutant VWF unhindered, which may have additional consequences, such as thrombocytopenia in patients with VWD type 2B, competition between mutant and normal VWF for platelet receptors, and the potential development of intestinal angiodysplasia. Most cases of VWD are caused by dominant‐negative mutations in VWF, and we hypothesize that diminishing expression of mutant VWF positively affects VWD phenotypes.

Objectives

To investigate allele‐specific inhibition of VWF by applying small interfering RNAs (siRNAs) targeting common single‐nucleotide polymorphisms (SNPs) in VWF. This approach allows allele‐specific knockdown irrespective of the mutations causing VWD.

Methods

Four SNPs with a high predicted heterozygosity within VWF were selected, and siRNAs were designed against both alleles of the four SNPs. siRNA efficiency, allele specificity and siRNA‐mediated phenotypic improvements were determined in VWF‐expressing HEK293 cells.

Results

Twelve siRNAs were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these siRNAs with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 siRNAs. We also demonstrated siRNA‐mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser.

Conclusions

Allele‐specific siRNAs are able to distinguish VWF alleles on the basis of one nucleotide variation, and are able to improve a severe multimerization defect caused by VWF p.Cys2773Ser. This holds promise for the therapeutic application of allele‐specific siRNAs in dominant‐negative VWD.