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1.  Phosphatidylserine Containing Liposomes Reduce Immunogenicity of Recombinant Human Factor VIII (rFVIII) in a Murine Model of Hemophilia A 
Journal of pharmaceutical sciences  2008;97(4):1386-1398.
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.
doi:10.1002/jps.21102
PMCID: PMC2574438  PMID: 17705286
hemophilia A; recombinant FVIII; immunogenicity; inhibitor antibodies; phosphatidylserine liposomes; protein delivery; protein formulation; lipids; immunology
2.  Immunogenicity and pharmacokinetic studies of recombinant Factor VIII containing lipid cochleates 
Drug delivery  2010;18(4):246-254.
Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15–30% of patients develop inhibitory antibodies against administered rFVIII which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previously, we developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. We hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. We investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as delayed release delivery vehicle with an input peak of rFVIII observed around 2 hrs post-injection. rFVIII associated with cochleates showed limited RES uptake and a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.
doi:10.3109/10717544.2010.536269
PMCID: PMC3068242  PMID: 21114461
3.  Effect of route of administration of human recombinant Factor VIII on its immunogenicity in Hemophilia A mice 
Journal of pharmaceutical sciences  2009;98(12):4480-4484.
Factor VIII is a multi-domain glycoprotein and is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes Hemophilia A, a bleeding disorder. Replacement using exogenous recombinant Factor VIII (FVIII) is the first line of therapy for Hemophilia A. Immunogenicity, the development of binding (total) and neutralizing (inhibitory) antibody against administered protein is a clinical complication of the therapy. There are several product related factors such as presence of aggregates, route and frequency of administration and glycosylation have been shown to contribute to immunogenicity. The effect of route of administration of Factor VIII on antibody development in Hemophilia A is not completely understood. Here we investigated the effect of route of administration (sc or iv) on immunogenicity in Hemophilia A mice. The total and inhibitory titers were determined using ELISA and modified Bethesda Assay respectively. The results indicated that sc is more immunogenic compared to iv route in terms of total antibody titer development (binding antibodies) but no significant differences in inhibitory titer levels could be established.
doi:10.1002/jps.21765
PMCID: PMC2796435  PMID: 19499565
Hemophilia A; Inhibitor development; Immunogenicity; Route of administration
4.  Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII 
The AAPS Journal  2009;11(3):424-431.
Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein–lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.
doi:10.1208/s12248-009-9119-y
PMCID: PMC2758112  PMID: 19499345
factor VIII; glycosylation; hemophilia A; immunogenicity; inhibitor development
5.  Lipid Binding Region (2303–2332) Is Involved in Aggregation of Recombinant Human FVIII (rFVIII) 
Journal of pharmaceutical sciences  2005;94(6):1288-1299.
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.
doi:10.1002/jps.20340
PMCID: PMC2583467  PMID: 15858858
recombinant human FVIII (rFVIII); physical instability; multi-domain; lipid-binding region; hemophilia A; inhibitor development
6.  Development and characterization of lipidic cochleate containing recombinant factor VIII 
Biochimica et biophysica acta  2007;1768(11):2890-2898.
Hemophilia A, a life threatening bleeding disorder is caused by deficiency of Factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII – containing cochleate cylinders, utilizing PS-Ca2+ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze – thawing and rate limiting diffusion studies revealed that small cochleate cylinders with a particles size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications.
doi:10.1016/j.bbamem.2007.08.001
PMCID: PMC2137893  PMID: 17936245
cochleate cylinders; B Domain Deleted recombinant factor VIII; epitope shielding; Laurdan; protein formulation; acrylamide quenching
7.  O-Phospho-L-Serine, Multi-functional Excipient for B Domain Deleted Recombinant Factor VIII 
The AAPS journal  2007;9(2):E251-E259.
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain, A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, ∼15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients’ plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid binding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free-vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDrFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.
doi:10.1208/aapsj0902028
PMCID: PMC2573386  PMID: 17907766
B domain deleted recombinant factor VIII; O-phospho-L-serine; protein formulation; excipient; physical stability; immunogenicity; inhibitor development
8.  O-phospho-L-serine, multi-functional excipient for B domain deleted recombinant factor VIII 
The AAPS Journal  2007;9(2):E251-E259.
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, ∼15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients' plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid bindinding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free-vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDRFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.
doi:10.1208/aapsj0902028
PMCID: PMC2573386  PMID: 17907766
B domain deleted recombinant factor VIII; O-phospho-L-serine; protein formulation; excipient; physical stability; immunogenicity; inhibitor development

Results 1-8 (8)