Phase I,randomized, double‐blind,placebo‐controlled,single‐dose escalation study of the recombinant factor VIIa variant BAY 86‐6150 in hemophilia

Summary. Background: BAY 86‐6150 is a new human recombinant factor VIIa variant developed for high procoagulant activity and longer action in people with hemophilia with inhibitors. Objectives: To investigate the safety, tolerability, pharmacodynamics, pharmacokinetics and immunogenicity of BAY 86‐6150 in non‐bleeding hemophilia subjects. Methods: The study included non‐bleeding men (18–65 years of age) with moderate or severe hemophilia A or B with or without inhibitors. Sixteen subjects were randomized 3 : 1 to four cohorts of escalating doses of BAY 86‐6150 (6.5, 20, 50 or 90 μg kg^?1 [n = 3 per cohort]) or placebo (n = 1 per cohort); an independent data‐monitoring committee reviewed previous cohort data before the next dose escalation. Blood sampling was performed predose and postdose; subjects were monitored for 50 days postdose. Results: At the tested doses, BAY 86‐6150 was not associated with clinically significant adverse events or dose‐limiting toxicities. BAY 86‐6150 pharmacokinetics exhibited a linear dose response, with a half‐life of 5–7 h. Subjects demonstrated consistent, dose‐dependent thrombin generation ex vivo in platelet‐poor plasma (PPP) (mean peak effect, 26–237 nm thrombin from 6.5 to 90 μg kg^?1). Peak thrombin levels over time paralleled BAY 86‐6150, with thrombin kinetics appearing to be slightly shorter; thus, circulating BAY 86‐6150 retained activity. There were corresponding decreases in activated partial thromboplastin and prothrombin times. No subject developed de novo anti‐BAY 86‐6150 neutralizing antibodies during the 50‐day follow‐up. Conclusions: In this first‐in‐human, multicenter, randomized, double‐blind, placebo‐controlled, single‐dose escalation study, BAY 86‐6150 was tolerated at the highest dose (90 μg kg^?1), with no safety concerns. Safety and efficacy will be further evaluated in phase II/III studies.     

Lentivirus‐mediated platelet gene therapy of murine hemophilia A with pre‐existing anti‐factor VIII immunity

Summary. Background: The development of inhibitory antibodies, referred to as inhibitors, against exogenous factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof‐of‐principle that platelet‐specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies. Objective: To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre‐existing inhibitors. Methods: Platelet FVIII expression in preimmunized FVIII^null mice was introduced by transplantation of lentivirus‐transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined with a chromogenic assay. The transgene copy number per cell was quantitated with real‐time PCR. Inhibitor titer was measured with the Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytically induced venous injury model. Integration sites were analyzed with linear amplification‐mediated PCR. Results: Therapeutic levels of platelet FVIII expression were sustained in the long term without evoking an anti‐FVIII memory response in the transduced preimmunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet FVIII expression resulting in phenotypic correction in preimmunized secondary and tertiary recipients. Conclusions: Lentivirus‐mediated platelet‐specific gene transfer improves hemostasis in mice with hemophilia A with pre‐existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high‐risk setting of pre‐existing inhibitory antibodies.     

Clinical guidelines and off‐license recombinant activated factor VII: content,use, and association with patient outcomes

Summary. Background: Recombinant activated factor VII (rFVIIa) is increasingly being used off‐license for treating critical bleeding. Guidelines may therefore be useful for improving processes and outcomes. Little is known regarding guidelines for off‐license rFVIIa or their association with patient outcomes. Objectives: To investigate the availability of hospital guidelines for off‐license rFVIIa use and the association between these guidelines and mortality. Methods: Data were extracted from the Haemostasis Registry, which collects all cases of off‐license rFVIIa use in participating institutions in Australia and New Zealand. Contributing hospitals were requested to supply a copy of the institutional guideline relating to off‐license rFVIIa administration. The characteristics of patients treated in accordance with all elements of the guidelines were compared with those of patients for who one or more guideline elements had been violated. The relationship between guideline‐directed treatment and 28‐day mortality was investigated using stepwise logistic regression. Results: Two thousand five hundred and fifty‐one patients in 75 hospitals were available for analysis. Of these hospitals, 58 provided a guideline for analysis. Patients complying with all guideline elements (n = 530) did not differ from patients receiving care that violated guidelines (n = 1035) regarding age, size of dose, or gender. Guideline‐directed treatment was not found to have an association with 28‐day mortality following logistic regression. Conclusions: Few patients are treated in accordance with the criteria of rFVIIa guidelines, despite their availability in the majority of hospitals. Moreover, 28‐day mortality does not appear to be associated with the use of guidelines in this patient group. Refinement of guidelines relating to the off‐license use of rFVIIa is therefore required.     

Bortezomib delays the onset of factor VIII inhibitors in experimental hemophilia A,but fails to eliminate established anti‐factor VIII IgG‐producing cells

Summary. Background: Replacement therapy with exogenous factor VIII to treat hemorrhages induces inhibitory anti‐FVIII antibodies in up to 30% of patients with hemophilia A. Current approaches to eradicate FVIII inhibitors using high‐dose FVIII injection protocols (immune tolerance induction) or anti‐CD20 depleting antibodies (Rituximab) demonstrate limited efficacy; they are extremely expensive and/or require stringent compliance from the patients. Objectives: To investigate whether the proteasome inhibitor bortezomib, which depletes plasmocytes, modulates the anti‐FVIII immune response in FVIII‐deficient mice. Methods and results: Preventive 4‐week treatment of naïve mice with bortezomib at the time of FVIII administration delayed the development of inhibitory anti‐FVIII IgG, and depleted plasma cells as well as different lymphoid cell subsets. Conversely, curative treatment of inhibitor‐positive mice for 10 weeks, along with FVIII administration, failed to eradicate FVIII inhibitors to extents that would be clinically relevant if achieved in patients. Accordingly, bortezomib did not eradicate anti‐FVIII IgG‐secreting plasmocytes that had homed to survival niches in the bone marrow, despite significant elimination of total plasma cells. Conclusions: The data suggest that strategies for the efficient reduction of anti‐FVIII IgG titers in patients with hemophilia A should rely on competition for survival niches for plasmocytes in the bone marrow rather than the mere use of proteasome inhibitors.     

Prevalence,biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one‐stage and chromogenic factor VIII activity

Summary. Background: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one‐stage assay. However, comparisons of these results with those of two‐stage assays can reveal discrepancies and suggest misdiagnosis. Patients/Methods: In this monocentric study, we measured FVIII:C with two methods (one‐stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. Results: Thirty‐six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. Conclusions: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of ‘inverse’ discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure–function relationships.     

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.

     

Recombinant factor XIII A‐subunit in a patient with factor XIII deficiency and recurrent pregnancy loss

Essentials

• Inherited factor XIII deficiency is a very rare bleeding disorder.
• We used recombinant factor XIII‐A in a pregnant patient with factor XIII‐A subunit deficiency.
• The patient had a successful pregnancy outcome with no pregnancy related complications.
• The dose of recombinant factor XIII‐A was minimized by using frequent trough level monitoring.

Summary

Inherited factor XIII deficiency is a very rare bleeding disorder, and is one of the causes of recurrent pregnancy loss. The use of plasma‐derived FXIII to improve pregnancy outcomes has been reported. We report a 26‐year‐old woman with FXIII A‐subunit (FXIII‐A) deficiency who was treated with recombinant FXIII‐A and had a successful pregnancy outcome with no pregnancy‐related complications. Our case illustrates that the dose of recombinant FXIII‐A can be minimized and adjusted on the basis of frequent trough level monitoring.