Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

Abstract

Abstract. Factor VIII (FVIII) is an important cofactor in blood coagulation cascade. It is a multidomain protein that consists of six domains, NH 2 -A1-A2-B-A3-C1-C2-COOH. The deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. Replacement therapy using recombinant FVIII (rFVIII) is the first line of therapy, but a major clinical complication is the development of inhibitory antibodies that abrogate the pharmacological activity of the administered protein. FVIII binds to anionic phospholipids (PL), such as phosphatidylinositol (PI), via lipid binding region within the C2 domain of FVIII. This lipid binding site not only consists of immunodominant epitopes but is also involved in von Willebrand factor binding that protects FVIII from degradation in vivo. Thus, we hypothesize that FVIII-PL complex will influence immunogenicity and catabolism of FVIII. The biophysical studies showed that PI binding did not alter conformation of the protein but improved intrinsic stability as measured by thermal denaturation studies. ELISA studies confirmed the involvement of the C2 domain in binding to PI containing lipid particles. PI binding prolonged the in vivo circulation time and reduced catabolism of FVIII in hemophilia A mice. FVIII-PI complex reduced inhibitor development in hemophilia A mice following intravenous and subcutaneous administration. The data suggest that PI binding reduces catabolism and immunogenicity of FVIII and has potential to be a useful therapeutic approach for hemophilia A.

PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice

“…Inhibitory titers, expressed in Bethesda units, are defined as the dilution at which 50% of FVIII activity is inhibited. Inhibitory titer data for free FVIII and FVIII-PI/PEG has been previously reported (31).…”

“…A thin lipid film composed of 50:50:5 molar ratio of DMPC/ PI/cholesterol was rehydrated with Tris buffer (25 mM Tris and 150 or 300 mM NaCl, pH=7.0) (31). In order to produce small unilamellar vesicles, multilamellar vesicles were extruded through double polycarbonate membranes of 80 nm pore size (GE Osmonics Labstore, Minnetonka, MN, USA) using a highpressure extruder (Northern Lipids Inc., Burnaby, BC, Canada) and then sterile-filtered through a 0.22-μm MillexTM-GP filter unit (Millipore Corporation, Bedford, MA, USA).…”

“…Noncompartmental analysis was performed using Phoenix WinNonlin v6.2 (Pharsight Corporation, Mountain View, CA, USA), and basic pharmacokinetic parameters including halflife (t 1/2 ), clearance (CL), volume of distribution (V), mean residence time (MRT), and area under the curve (AUC) were generated. Comparisons were made to the pharmacokinetics of free FVIII and FVIII-PI previously obtained under identical experimental conditions (31).…”

“…Immunizations were administered subcutaneously, and it has been shown that inhibitory titers to FVIII generated via this route are comparable to those following i.v. administration (31,44). The results obtained from immunogenicity studies are summarized in Table III.…”

“…PEGylation of FVIII-PS liposomes resulted in a further reduction of immune response compared to the un-PEGylated formulation but provided only a modest improvement in the pharmacokinetic profile (30). Replacement of PS with phosphatidylinositol (PI) in FVIII containing lipidic particles provided a significant improvement in the half-life and avoided the rapid RES clearance observed with the previous formulations (31). Both PS and PI interact with the amino acid 2303-2332 lipid binding C2 domain of FVIII, but PI also associates with the A2 domain and FVIII is believed to penetrate deeper into the lipid particle (32)(33)(34).…”