Nonlinear pharmacokinetics of factor VIII and its phosphatidylinositol lipidic complex in hemophilia A mice.

Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade and its deficiency or dysfunction causes hemophilia A (HA), a bleeding disorder. Replacement with recombinant FVIII is limited by a short half-life and the development of inhibitory antibodies. A phosphatidylinositol (PI) containing lipid nanoparticle was developed that, when associated with FVIII, reduces immunogenicity and prolongs circulation of the therapeutic protein in HA mice. A multiple dose level pharmacokinetic (PK) study of human free FVIII and its FVIII-PI complex over a clinically relevant range of doses (20, 40 and 200 IU/kg) was conducted in HA mice to investigate linearity of the PK and to determine if the reduced catabolism of FVIII following association with PI particles, previously only observed in the terminal phase following 400 IU/kg, could be extendable over a range of doses. The findings suggest that the disposition of FVIII is best characterized by a two-compartment model with saturable Michaelis-Menten elimination. Spontaneous complexation of FVIII with PI particles significantly increases plasma survival of the protein at 20 and 40 IU/kg doses. Human simulations at 40 IU/kg project an increase in the terminal half-life and the time to reach a minimum therapeutic threshold of 0.01 IU/ml of 5.4 h and 40 h, respectively, compared with free FVIII. Formulation with PI containing lipid particles may represent a viable delivery strategy for improving FVIII therapy.

Permeability of surface-modified polyamidoamine (PAMAM) dendrimers across Caco-2 cell monolayers.

Aim of this study was to prepare polyamine-conjugated PAMAM dendrimers and study their permeability across Caco-2 cell monolayers. Polyamines, namely, arginine and ornithine were conjugated to the amine terminals of the G4 PAMAM dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P(app)) for the PAMAM dendrimers increased by conjugating the dendrimers with both of the polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. The correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with both the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of the polyamine-conjugated dendrimers was evaluated in Caco-2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were slightly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as drug carriers to increase the oral absorption of drugs.

Allometry of factor VIII and informed scaling of next-generation therapeutic proteins.

Allometric scaling has been applied to the pharmacokinetics (PK) of factor VIII (FVIII), but published relationships are based on relatively small subsets of available data. Numerous next-generation forms of FVIII are being developed (e.g., Fc fusion, PEGylated, and liposomal formulations) and traditional PK scaling of these products would not incorporate the wealth of existing knowledge for current FVIII therapy in humans. We conducted a meta-analysis and developed allometric relationships of FVIII from over 100 PK studies collected from literature. Normalized Wajima curves were used to relate mean FVIII profiles between species. An "informed scaling" approach was derived for predicting first-in-human PK parameters and demonstrated with a case study for an Fc fusion FVIII. NCA values for FVIII PK were well described by the allometric equations CL = 6.59 W(0.85) and V(ss) = 65.0 W(0.97). A subset of studies characterized by two-compartment modeling showed strong linearity in scaling of total clearance (CL) and central volume, but more variability in distributional CL and peripheral volume. Wajima curves for FVIII superimposed across species and the disposition of Fc fusion FVIII in humans was well predicted by "informed scaling." This approach might be generally applicable for predicting human PK of next-generational therapeutics.

Phosphatidylinositol induces fluid phase formation and packing defects in phosphatidylcholine model membranes.

Liposomes consisted of phosphatidylinositol (PI) and phosphatidylcholine (PC) have been utilized as delivery vehicle for drugs and proteins. In the present work, we studied the effect of soy PI on physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes such as phase state of lipid bilayer, lipid packing and phase properties using multiple orthogonal biophysical techniques. The 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan) fluorescence studies showed that presence of PI induces the formation of fluid phases in DMPC. Differential scanning calorimetry (DSC), temperature dependent fluorescence anisotropy measurements, and generalized polarization values for Laurdan showed that the presence of as low as 10mol% of PI induces substantial broadening and shift to lower temperature of phase transition of DMPC. The fluorescence emission intensity of DPH labeled, PI containing DMPC lipid bilayer decreased possibly due to deeper penetration of water molecules in lipid bilayer. In order to further delineate the effect of PI on the physico chemical properties of DMPC is due to either significant hydrophobic mismatch between the acyl chains of the DMPC and that of soy PI or due to the inositol head group, we systematically replaced soy PI with PC species of similar acyl chain composition (DPPC and 18:2 (Cis) PC) or with diacylglycerol (DAG), respectively. The anisotropy of PC membrane containing soy PI showed largest fluidity change compared to other compositions. The data suggests that addition of PI alters structure and dynamics of DMPC bilayer in that it promotes deeper water penetration in the bilayer, induces fluid phase characteristics and causes lipid packing defects that involve its inositol head group.

Native-like aggregates of factor VIII are immunogenic in von Willebrand factor deficient and hemophilia a mice.

The administration of recombinant factor VIII (FVIII) is the first-line therapy for hemophilia A (HA), but 25%-35% of patients develop an inhibitory antibody response. In general, the presence of aggregates contributes to unwanted immunogenic responses against therapeutic proteins. FVIII has been shown to form both native-like and nonnative aggregates. Previously, we showed that nonnative aggregates of FVIII are less immunogenic than the native protein. Here, we investigated the effect of native-like aggregates of FVIII on immunogenicity in HA and von Willebrand factor knockout (vWF(-/-)) mice. Mice immunized with native-like aggregates showed significantly higher inhibitory antibody titers than animals that received native FVIII. Following restimulation in vitro with native FVIII, the activation of CD4+ T-cells isolated from mice immunized with native-like aggregates is approximately fourfold higher than mice immunized with the native protein. Furthermore, this is associated with increases in the secretion of proinflammatory cytokines IL-6 and IL-17 in the native-like aggregate treatment group. The results indicate that the native-like aggregates of FVIII are more immunogenic than native FVIII for both the B-cell and the T-cell responses.

Delivery of therapeutic proteins.

The safety and efficacy of protein therapeutics are limited by three interrelated pharmaceutical issues, in vitro and in vivo instability, immunogenicity and shorter half-lives. Novel drug modifications for overcoming these issues are under investigation and include covalent attachment of poly(ethylene glycol) (PEG), polysialic acid, or glycolic acid, as well as developing new formulations containing nanoparticulate or colloidal systems (e.g., liposomes, polymeric microspheres, polymeric nanoparticles). Such strategies have the potential to develop as next generation protein therapeutics. This review includes a general discussion on these delivery approaches.

Use of a folding model and in situ spectroscopic techniques for rational formulation development and stability testing of monoclonal antibody therapeutics.

Aggregation is a critical issue that hampers the development of monoclonal antibody therapeutics (Mabs). Traditionally, aggregation is considered a process in which native forms of proteins are transformed into an unstable highly associated form through an intermediate formation step. Here we describe the unfolding of an antiCD40 antibody using a folding model based on Lumry-Eyring nucleated polymerization (LENP) model. This model captures several experimental features of the thermal unfolding of this protein as studied by common in situ biophysical techniques such as circular dichroism, fluorescence spectroscopy, and turbidity measurements. According to this model, the unfolding and aggregation of the antiCD40 antibody is determined by several distinct steps that include conformational change(s) to generate aggregation prone states, reversible oligomer formation, nucleation and growth as well as their kinetics, and the formation of higher order assemblies/aggregates. Furthermore, the loss of monomer is controlled by both thermodynamic (equilibrium unfolding) and kinetic determinants of the unfolding process. This approach captures both of these rate-limiting steps. It can be concluded that this approach is sensitive to formulation conditions such as protein concentration, changes in buffer conditions, and temperature stress. The potential use of this approach in formulation development and stability testing of Mabs is discussed.

Transport of surface engineered polyamidoamine (PAMAM) dendrimers across IPEC-J2 cell monolayers.

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM(G4) dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P(app)) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.