Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs.

Human serum albumin (HSA) is a natural carrier protein possessing multiple ligand binding sites with a plasma half-life ~19days, facilitated by interaction with the human neonatal Fc receptor (FcRn), that promotes it as a highly attractive drug delivery technology. A lack of adequate rodent models, however, is a major challenge in the preclinical development of albumin-linked therapeutics. This work describes the first double transgenic mouse model bearing both human FcRn and HSA genes (hFcRn(+/+), hAlb(+/+)) under the control of an endogenous promoter. Human FcRn was shown by immunohistochemical and qPCR analysis to be ubiquitously expressed in the major organs. Physiological levels of HSA were detected in the blood that exhibited similar FcRn binding kinetics to recombinant or human serum-derived HSA. The circulatory half-life (t1/2) was shown to be dependent on FcRn binding affinity that increased from low affinity (t1/2 29h), to wild type (t1/2 50h), to high affinity (t1/2 80h) variants, that validates the application of the model for optimizing the pharmacokinetics of drug carriers who's circulatory half-life is dependent in some manner upon interaction with endogenous FcRn. This study presents a novel mouse model that better mimics the human physiological conditions and, thus, has potential wide applications in the development of albumin-linked drugs or conventional drugs whose action is influenced by reversible binding to endogenous HSA.

Electrochemical analysis of the fibrillation of Parkinson's disease α-synuclein.

Amyloid formation of proteins and peptides is an important biomedical and biotechnological problem, intensively studied and yet not fully understood. In this context, the development of fast and reliable methods for real-time monitoring of protein misfolding is of particular importance for unambiguous establishment of disease-, drug- and environmentally induced mechanisms of protein aggregation. Here we show that the extent of aggregation of α-synuclein (αSN), involved in Parkinson's disease and other neurodegenerative disorders, can be electrochemically monitored by oxidizing tyrosine (Tyr) residues surface-exposed in monomeric αSN and buried in fibrillated αSN adsorbed onto graphite electrodes. Adsorption of αSN, analyzed through the Tyr electrochemistry, followed the Langmuir adsorption isotherm. The degree of electrooxidation of Tyr in αSN decreased upon protein fibrillation and correlated with the extent of αSN aggregation determined by the spectroscopic analysis of the fibrillation process. Minor changes in the adsorption state of αSN were followed through the shift of the Tyr oxidation potential, consistent with the compact and less-compact/unfolded conformation of αSN. Our results allow reliable electroanalysis of the extent of αSN fibrillation in vitro and offer an efficient tool for future in vivo monitoring of the protein conformational state.