Monoclonal antibody (mAb) engineering that optimizes binding to receptors present on brain vascular endothelial cells has enabled them to cross through the blood-brain barrier (BBB) and access the brain parenchyma to treat neurological diseases. However, once in the brain the extent to which receptor-mediated reverse transcytosis clears mAb from the brain is unknown. The aim of this study was to determine the contribution of the neonatal Fc-receptor (FcRn) in rat brain efflux employing two different in vivo drug delivery models. Two mAb variants with substantially different affinities to FcRn, and no known neuronal targets, (IgG1 N434A and H435A) were administered to rats via intranasal-to-central nervous system (CNS) and intra-cranial dosing techniques. Levels of full-length IgG were quantified in serum and brain hemispheres by a sensitive enzyme-linked immunosorbent assay (ELISA). Following intra-nasal delivery, low cerebral hemisphere levels of variants were obtained at 20min, with a trend towards faster clearance of the high FcRn binder (N434A); however, the relatively higher serum levels confounded analysis of brain FcRn contribution to efflux. Using stereotaxic coordinates, we optimized the timing and dosing regimen for injection of mAb into the cortex. Levels of N434A, but not H435A, decreased in the cerebral hemispheres following bilateral injection into the rat cortex and higher levels of N434A were detected in serum compared to H435A after 24h. Immunohistochemical staining of human IgG1 in sections of cortex was consistent with these results, illustrating relatively less intense immunostaining in N434A than H435A dosed animals. Using two in vivo methods with direct cranial administration, we conclude that FcRn plays an important role in efflux of IgG from the rat brain.

The past decade has seen a number of significant changes in identifying higher quality lead compounds earlier in the drug discovery process. Cell-based assay technologies yielding high-content information have emerged to achieve this goal. Although most of these systems are based on fluorescence detection, this article describes the development and application of an innovative cellular assay technology based on radio frequency spectrometry and bioimpedance measurements. Using this technique, the authors have discovered a link between cellular bioimpedance changes and receptor-mediated signal transduction events. By performing dielectric spectroscopy of cells across a spectrum of frequencies (1 KHz to 110 MHz), a series of receptorspecific, frequency-dependent impedance patterns is collected. These raw data patterns are used to determine the identity of the cellular receptor-signaling pathway being tested and to quantify stimulation endpoints and kinetics. The authors describe the application of this technology to the analysis of ligand-induced cellular responses mediated by the 3 major classes of Gprotein-coupled receptors (GPCRs) and protein tyrosine kinase receptors. This single assay platform can be used with ease to monitor G s , G i , and G q GPCRs without the need for chimeric or promiscuous G-proteins, fluorophors, or tagged proteins. In contrast to other methods of monitoring cellular signal transduction, this approach provides high information content in a sim-