This could explain the lack of a clear difference between the two variants following unilateral administration; therefore, the variants were dosed bilaterally. The total brain dose of 2.4 µg was the same as in the previous study, but a 1.2 µg (0.6 µL) dose of either N434A or H435A was administered bilaterally. Serum levels were measured at 5 min, 4, and 24 h and rats were allowed to recover from anesthesia immediately after surgery. After 24 h, N434A serum levels reached 25.9±5.2 ng/mL which was significantly greater (P<0.001) than the serum levels of the low FcRn binding variant, H435A, at 6.6±0.6 ng/mL.

The rate of efflux of N434A was calculated to be three times greater MI-773 concentration than H435A ( Fig. 4A). In a separate group of animals, brain hemispheres were excised 5 min after either N434A or H435A antibody administration. Levels of N434A detected at 5 min were 2.2±0.5 µg/g

of tissue (average mass of the hemispheres was 1.0 g) which was the expected range after administration. After 24 h, levels of N434A in the hemispheres decreased by 48%. Levels of the non-FcRn binding mAb, H435A, did not change over the same time period (Fig. 4B). In order to visualize the presence of human IgG immunohistochemically in the rat brain, three animals were dosed either N434A or H435A bilaterally into the SiFl region of the cortex similar to the previous experiment. After 24 h rats were perfused and brains prepared for immunohistochemistry LGK-974 cell line using an anti-human Fc rabbit polyclonal in thin (5 µm) brain sections. Brains were processed for routine histology (hematoxylin and eosin and Luxol fast blue), as well as human IgG and anti-rabbit IgG negative controls. At 24 h after dosing both N434A and H435A mAb variants were visualized in brain tissue (Fig. 5). There was a range of staining intensity visualized in sections at an equivalent coronal plane in the region of the somatosensory cortex in pyramidal and supporting cells that was localized to the cortex or spread into the corpus callosum region. The staining intensity of N434A (Fig. 5A) in the cortex was consistently

less than brains injected with clonidine H435A (Fig. 5B). There was no specific staining detected in PBS microinjected brains (Fig. 5C) or mAb injected brains incubated in serum (Fig. 5D). Semi-quantitative immunostaining scores supported the impression that less of the FcRn binding variant was visualized in brain after 24 h (HA=7.0±1.4 vs. NA=4.4±1.7 of a total of 12.0 maximum staining; P=0.3 by unpaired t-test), which is consistent with greater efflux of the FcRn binding mAb variant. This study used two in vivo drug delivery models in rats, intranasal-to-CNS and direct intra-cranial dosing, to determine the contribution of FcRn to IgG receptor-dependent reverse-transcytosis (efflux) from the brain parenchyma to the circulation.