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.

     

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.     

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.

     

Novel insights into the development of atherosclerosis in hemophilia A mouse models

Summary. Background: Cardiovascular diseases in aging people with hemophilia (PWH) represent a growing concern. The underlying hypocoagulability probably provides a protective effect against acute thrombus formation, but the limited data available show no preventive effect against the development of atherogenesis in PWH. Atherosclerosis‐prone mice are attractive tools for the study of atherosclerosis development, and may provide insights into disease progression in PWH. Methods: Severe hemophilia A (factor VIII‐deficient [FVIII^o]) mice were crossed with mice lacking apolipoprotein E (ApoE^?/?) or mice lacking the LDL receptor (LDLR^?/?), and then compared to hemostatically normal littermate controls. After mice had received atherogenic diets for 8, 22 or 37 weeks, atherosclerotic lesion size and phenotypic characterization were analyzed in the aortic sinus and whole aortas. Results: ApoE^?/?/FVIII^o mice showed a time‐dependent protective effect against the development of atherosclerosis, beginning after 22 diet‐weeks and persisting to 37 diet‐weeks in both the aorta sinus and whole aorta as compared with ApoE^?/? mice. Notably, the FVIII deficiency did not influence the progression of atherosclerosis in the FVIII^o/LDLR^?/? model as compared with controls at early or late time points. Conclusions: Hypocoagulability ameliorates vascular disease in the ApoE‐deficient model in a lipid‐independent manner. Interestingly, FVIII deficiency did not affect the development of atherosclerosis in LDLR^?/? mice. In contrast to the ApoE model, the LDLR model resembles the lipid profile that is commonly observed in humans with atherosclerosis. These findings, to a certain extent, support the notion of atherosclerosis development in the complete absence of FVIII.     

Characterization of duplication breakpoints in the factor VIII gene

See also Goodeve AC. Another step towards understanding hemophilia A molecular pathogenesis. This issue, pp 2693–5. Summary. Background: Hemophilia A is caused by a wide spectrum of different mutations in the factor (F)VIII gene (F8), leading to deficiencies in coagulation FVIII activity and thus resulting in an inefficient blood clotting cascade. Large duplications comprising whole exons of F8 have been published for only a few cases so far. Results: In the current study, we characterized the exact breakpoints for a total of 10 exon‐spanning duplications of F8, including six novel duplications in seven unrelated patients. Seven breakpoints were located within long interspersed nuclear elements (LINEs), whereas short interspersed nuclear elements (SINEs) of the Alu‐repeat type were observed at both breakpoint sites in four of the 10 duplications. At three breakpoints, microhomologies of 2 bp and 3 bp each could be identified. Conclusions: Duplication breakpoints in F8 were shown to be located in repetitive elements, especially SINEs or LINEs, but also in unique sequences. In addition, microhomologies, particular genomic features or sequence motifs, contribute to the duplication formation mechanisms.