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The replacement therapy using recombinant human FVIII (rFVIII) is the first line of therapy for hemophilia A. Approximately 15–30% of the patients develop inhibitory antibodies. Recently, we reported that liposomes composed of phosphatidylserine (PS) could reduce the immunogenicity of rFVIII. However, PS containing liposomal-rFVIII is likely to reduce the systemic exposure and efficacy of FVIII due to rapid uptake of the PS containing liposomes by the reticuloendothelial system (RES). Here, we investigated whether phosphatidylserine (PS) liposomes containing Polyethylene glycol (PEG) (PEGylated), could reduce the immunogenicity of rFVIII and reverse the reduction in systemic exposure of rFVIII. Animals given PEGylated liposomal-rFVIII had lower total and inhibitory anti-rFVIII antibody titers, compared to animals treated with rFVIII alone. The mean stimulation index of CD4+ T-cells from animals given PEGylated liposomal-rFVIII also was lower than for animals that were given rFVIII alone. Pharmacokinetic studies following intravenous dosing indicated that the systemic exposure (area under the activity curve, AUAC0–24h) of PEGylated liposomal-rFVIII was ∼59 IU/mL × h and significantly higher than that of non-PEGylated liposomal-rFVIII (AUAC0–24h ∼ 36 IU/mL × h). Based on these studies, we speculate that PEGylated PS-containing liposomal rFVIII may improve efficacy of rFVIII. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3753–3764, 2008 [ABSTRACT FROM AUTHOR]

Factor VIII (FVIII) is a multidomain protein that is deficient in hemophilia A, a clinically important bleeding disorder. Replacement therapy using recombinant human FVIII (rFVIII) is the main therapy. However, approximately 15–30% of patients develop inhibitory antibodies that neutralize rFVIII activity. Antibodies to epitopes in C2 domain, which is involved in FVIII binding to phospholipids, are highly prevalent. Here, we investigated the effect of phosphatidylserine (PS)-containing liposomes, which bind to C2 domain with high affinity and specificity, upon the immunogenicity of rFVIII. Circular dichroism studies showed that PS-containing liposomes interfered with aggregation of rFVIII. Immunogenicity of free- versus liposomal-rFVIII was evaluated in a murine model of hemophilia A. Animals treated with s.c. injections of liposomal-rFVIII had lower total- and inhibitory titers, compared to animals treated with rFVIII alone. Antigen processing by proteolytic enzymes was reduced in the presence of liposomes. Animals treated with s.c. injections of liposomal-rFVIII showed a significant increase in rFVIII plasma concentration compared to animals that received rFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between PS-containing bilayers and rFVIII may provide a basis for designing lipidic complexes that improve the stability, reduce the immunogenicity of rFVIII formulations, and permit administration by s.c. route. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1386–1398, 2008 [ABSTRACT FROM AUTHOR]

The physical phenomenon of aggregation can have profound impact on the stability of therapeutic proteins. This study focuses on the aggregation behavior of recombinant human FVIII (rFVIII), a multi-domain protein used as the first line of therapy for hemophilia A, a bleeding disorder caused by the deficiency or dysfunction of factor VIII (FVIII). Thermal denaturation of rFVIII was investigated using circular dichroism (CD) spectroscopy and size exclusion chromatography (SEC). The dependence of unfolding on heating rate indicated that the thermal denaturation of the protein was at least partly under kinetic control. The data was interpreted in terms of a simple two-state kinetic model, $N(Native)\buildrel k \over\longrightarrow A({\rm Aggregated})$, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation. Analysis of the data in terms of the above scheme suggested that under the experimental conditions used in this study, the rate-controlling step in the aggregation of rFVIII may be a unimolecular reaction involving conformational changes. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2023–2029, 2005 [ABSTRACT FROM AUTHOR]

Factor VIII (FVIII) is a multi-domain protein that is important in the clotting cascade. Its deficiency causes Hemophilia A, a bleeding disorder. The unfolding of protein domains can lead to physical instability such as aggregation, and hinder their use in replacement therapy. It has been shown that the aggregation of rFVIIII is initiated by small fluctuations in the protein's tertiary structure (Grillo et al., 2001, Biochemistry 40:586–595). We have investigated the domain(s) involved in the initiation of aggregation using circular dichroism (CD), size exclusion chromatography (SEC), fluorescence anisotropy, domain specific antibody binding, and clotting activity studies. The studies indicated that aggregation may be initiated as a result of conformational change in the C2 domain encompassing the lipid-binding region (2303–2332). The presence of O-phospho-L-Serine (OPLS), which binds to the lipid-binding region of FVIII, prevented aggregation of the protein. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1288–1299, 2005 [ABSTRACT FROM AUTHOR]