Phosphatidylinositol reduces the immunogenicity and clearance of human recombinant factor VIII in hemophilia a mice

Factor VIII (FVIII), an important cofactor in blood coagulation cascade, is a multidomain protein that consists of six domains, NH2-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 in about one third of the patients. In addition, fast clearance of FVIII in vivo adds another barrier to the clinical management of the disease.;The overall goal of this dissertation was to develop FVIII preparations that improve therapeutic efficacy of rFVIII by reducing the immunogenicity and enhancing the circulation half-life of the protein. Our approach to address the current clinical challenges of rFVIII is through the rational development of multi-functional lipid-based FVIII formulations that are less immunogenic and long acting in vivo, thereby reducing the frequency of administration. We rationally developed lipid based FVIII formulation by modifying liposomes through different parameters that are known to affect in vivo behavior of liposomes, including liposome membrane fluidity, particle size and the presence of hydrophilic polymers (surface hydration). We hypothesize that by associating FVIII with phosphatidylinositol (PI)-containing lipidic particles via the C2 domain would (1) interfere with FVIII aggregation process thereby improving the intrinsic stability of the protein, (2) prolong the circulation half-life of FVIII, and (3) reduce FVIII immunogenicity.;We successfully associated FVIII with PI-containing lipidic particles with high efficiency (Chapter 2). Based on the biochemical studies, we confirmed the involvement of the C2 domain in binding to PI-containing lipidic particles and showed the participation of other FVIII domains in PI binding. This observation suggested that a substantial surface area of the protein was buried in the PI particles. In vitro activity experiments and biophysical studies showed that the binding between FVIII and PI particles did not interfere with the activity of the protein nor does it alter the conformation of the protein. Further, the intrinsic stability of FVIII was improved when associated with PI particles. We hypothesized that high loading efficiency of FVIII in PI particles might be due to lipid packing defects in the membrane bilayer of PI particles, which allowed deeper penetration of FVIII molecule into the bilayer. In order to test this lipid packing defects, we conducted a series of biophysical characterization on PI particles using several biophysical techniques (Chapter 3). Our results showed that PI induces the formation of liquid crystalline (LC) phase in DMPC membrane and causes lipid packing defects.;The effect of PI binding on longer acting properties of FVIII was investigated in FVIII-knockout murine model (Chapter 4). The circulation half-life of FVIII associated with PI was ∼4 fold higher than that observed for free FVIII suggesting that PI binding prolonged the in vivo circulation time and reduced catabolism of FVIII. The prolongation of FVIII circulation half-life in the presence of PI is clinically relevant as shown in the efficacy studies. Both FVIII inhibitory antibody titers and total antibody titers were significantly reduced in hemophilia A mice that received FVIII-PI complex suggesting that FVIII is less immunogenic when bound to PI particles (Chapter 5). This reduction in FVIII immunogenicity by FVIII-PI is immunologically significant as FVIII bound to PI reduced the proliferation of FVIII-specific T-cells. Considering that PEGylated liposomes have been shown to improve biopharmaceutical properties of rFVIII in both preclinical and clinical studies, we continued to explore FVIII-PI formulation by incorporating polyethylene glyco (PEG) conjugated lipid into PI-containing lipidic particles (Chapter 6). The circulation half-life of FVIII associated with PI/PEG was about 3 fold higher than that observed for FVIII-PI, and 13 fold higher than that observed for free FVIII in hemophilia A mice. Moreover, PI/PEG not only reduced overall anti-FVIII antibody titers by about 3 fold, but it also significantly reduced the titer of antibodies that abrogate the activity of the protein.;In summary, this work has succeeded in developing novel FVIII preparation using lipid based drug delivery approach which improved the therapeutic efficacy for rFVIII.