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. KeywordsProtein delivery; PEGylation; Liposomes; hyperglycosylation; Poly(lactic/glycolic) acid INTRODUCTIONSince the late 20 th century numerous therapeutic proteins and peptides have emerged in the market. PHARMA 2010 reported that biotech products accounted for more than 35% of the 37 new active substances launched in 2001.1 In 2007, global biotech drug sales grew at twice the rate of traditional small molecule drugs (12.5% vs 6.4%) with total revenues of $75 billion US. Biotech drugs accounted for one fifth of all blockbuster drugs in the market as of 2008.2 From a therapeutic perspective, proteins offer the distinct advantage of specific mechanisms of action and are highly potent. Despite these advantages, biotech products must overcome the hurdles posed by high molecular weight, short half-lives, instability, and immunogenicity. Several strategies have been evaluated in an effort to improve the current limitations of therapeutic peptides and proteins in the creation of so called "second generation" protein therapeutics. Most efforts center around one of two approaches-either a change in the agent itself (e.g. mutations in protein structure or covalent attachment of moieties) or by a change in formulation.3 In contrast to modifying the protein structure, covalent chemical attachment of compounds such as poly(ethylene glycol) (PEG) or polysialic acid (PSA) to therapeutic protein represent a relatively new approach. Drug formulation systems, such as liposomes, polymeric microspheres, and polymeric nanoparticles, are another means to help overcome the current limitations of protein therapeutics.4 , 5The intent of this review is to provide a general discussion of approaches being applied to improve safety and efficacy of protein therapeutics. This includes the areas of PEGylation, PEGYLATIONThe conjugation of polymers to proteins had been in practice since the 1950s, but it was the development of PEGylation that provided the real breakthrough in enhancing the pharmaceutical properties of proteins and peptides in a viable manner 6 PEGylation, the covalent attachment of PEG moieties to a therapeutic agent, was first reported in the 1970s. 7,8 Experiments attempting to improve delivery aspects via PEGylation found not only the intended benefits, but overall enhancement of stability, pharmacokinetics, and therapeutic utility of molecules. [9][10][11][1...
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 apicalto-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.
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 non-native aggregates. Previously, we showed that non-native aggregates of FVIII are less immunogenic compared to 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 compared to animals that received native FVIII. Following re-stimulation in vitro with native FVIII, the activation of CD4+ T cells isolated from mice immunized with native-like aggregates is ~4 fold higher than mice immunized with the native protein. Furthermore, this is associated with increases in the secretion of pro-inflammatory 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 T cell responses.
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 10 mol% 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.
Hemophilia A is a bleeding disorder caused by the deficiency of Factor VIII (FVIII)(1). In circulation, FVIII binds to its carrier protein vWF with high affinity (Kd < 0.5nM) and this strong non-covalent interaction is critical for the normal half life and circulating plasma concentration of FVIII (2 -3). The complex protects FVIII from proteolytic degradation by proteases including activated protein C and prevents cellular uptake by LRP (2). The noncovalent interaction of vWF with FVIII involves amino acids 1672-1689 within the A3 domain and 2303-2332 of the C2 domain of . LRP binds to FVIII via A2, A3 and C2 domains (5 -6). The monoclonal antibody ESH4, directed against residues 2303-2332 of the C2 domain of FVIII, inhibits binding to both vWF and LRP (5). This implies that vWF and LRP binding sites are in close proximity and/or overlap. This same region (2303-2332) also mediates the interaction of FVIII with phosphatidylserine (PS) on platelet membranes (7). Recent evidences also suggest the participation of C1 domain of FVIII in lipid binding (8 -9). FVIII also binds to other anionic lipids including phosphatidylinositol (PI), but the specificity and affinity is highest for PS compared to other anionic lipids (10). Based on these studies it is interesting to note that within C2 domain the binding site for three macromolecular interactions of vWF, LRP and phospholipids either overlap or in close proximity ( Figure 1a). We have demonstrated previously that FVIII-PS and FVIII-PI complex reduced inhibitor development (11 -12), suggesting therapeutic potential of FVIII-lipid complex. However, the catabolism of FVIIIlipid complex is not completely understood and could be complicated due to overlapping or proximal binding sites and comparable binding affinities in nM range for both phospholipid and vWF. Further phospholipid binding can sterically block macromolecular interaction involving C1 and C2 domain of FVIII. In order to delineate the role of vWF on catabolism of FVIII-lipid complex, pharmacokinetic studies of FVIII in the presence and in the absence of PS and PI, in Hemophilia A (HA) and vWF −/− mice were carried out. Our data suggests that FVIII-lipid complex showed an improved pharmacokinetic profile in both HA mice as well as in vWF −/− mice. All animal handling was performed in accordance with the guidelines of institutional animal care and use committees (IACUC) at the University at Buffalo.FVIII-PI and FVIII-PS complexes were prepared as described previously (11). Pharmacokinetic studies and analysis were carried out as described previously (13 -14). Statistical Analysis was conducted using Minitab 15 (Minitab Inc, State College, PA). Statistical differences (p<0.05) were tested using the Student independent t-test and one-way ANOVA followed by Dunnet's post-hoc multiple comparison test. Systemic exposures were compared using Bailer-Satterwaite method(15). In order to establish the role of vWF in FVIII catabolism and to obtain relevant pharmacokinetic parameters, pharmacokinetic studies of FVIII were ...
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