Seminars in Pharmaceutical Sciences
Ph.D. Thesis Defense
Pharmaceutical Sciences, UW-Madison
(under the supervision of Prof. Robert Thorne)
Delivering antibodies to the brain: distribution, transport mechanisms, and dose-response
following intranasal or systemic administration
Antibody-based therapeutics have gained significant momentum as potential treatments for several central nervous system (CNS) disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and brain cancer, among others. However, drug delivery to the CNS for antibodies and other macromolecules has thus far proven challenging, due in large part to the blood-brain barrier (BBB) and blood-cerebrospinal fluid barriers (BCSFBs) that greatly restrict transport of administered molecules from the systemic circulation into the CNS.
This work investigated the potential of intranasal delivery to rapidly and non-invasively target antibodies to the CNS. Using complementary fluorescence and radiometric techniques, we demonstrated that full length immunoglobulin G (IgG) antibodies can access perineural and perivascular distribution pathways associated with the olfactory and trigeminal nerves to reach CNS entry sites (olfactory bulbs and brainstem respectively), and the perivascular space (PVS) associated with cerebral blood vessels to subsequently distribute within the brain parenchyma. The PVS contains a mixture of CSF and interstitial fluid (ISF) that is under convective or dispersive flow driven by the pulsation of arteries. Transport in this compartment is likely to scale with brain size across different species. The ability of intranasally administered IgG to access the perivascular compartment is therefore highly relevant for potential clinical translation. We also reported two major factors that may affect the capacity of antibodies to access or exit the PVS. First, entry into the PVS is size-dependent and smaller antibody fragments (e.g., Fab fragments) can access the PVS to a greater extent compared to full length IgG. Second – Fc gamma receptors are expressed in brain regions surrounding the PVS and may potentially slow down or impede IgG diffusion out of the PVS and into the brain parenchyma.We also provide a detailed comparison of antibody delivery to the CNS following intranasal versus intra-arterial administration with respect to tissue distribution and dose-response. We observed that for IgG doses resulting in similar blood levels following intranasal or intra-arterial delivery, the levels in the CNS were significantly higher with intranasal administration. Intranasal delivery also showed a more favorable dose-response compared to intra-arterial delivery. Overall, our findings suggest that intranasal application of IgG can rapidly achieve therapeutically relevant CNS concentrations (mid picomolar to low nanomolar range) by accessing pathways and mechanisms that bypass the BBB and BCSFBs and are not easily saturated.
Additionally, we investigated the intactness of intranasally applied IgG in the brain providing implications for the biochemical conditions necessary for IgG intactness in vivo. Using non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, we demonstrated that the brain soluble protein fraction following intranasal delivery of radiolabeled IgG yielded distinct heavy and light chain radiolabeled protein bands. Our results show that disulfide bond reduction of full length IgG is likely a consequence of tissue processing, as well as some degree of potential antibody catabolism in vivo. Importantly, brain tissue homogenization in the presence of an oxidizing agent added to the lysis buffer preserved some signal associated with intact IgG in the brain following intranasal delivery. In conclusion, our results are consistent with successful delivery of IgG to the brain.
Finally, we hypothesized that delivering drugs to nasal regions that have a lower vascularity and/or permeability may allow more drug to access the extracellular cranial nerve associated pathways and therefore favor delivery to the brain. However, vascularities and relative vascular permeabilities of the different nasal mucosal sites were previously largely unknown. We determined that the relative vascular permeability is significantly greater in nasal respiratory regions than in olfactory regions. Mean capillary density (i.e., vascularity) in the nasal mucosa was also approximately 5-fold higher in nasal respiratory regions than in olfactory regions. Applying capillary pore theory and a normalization strategy to our experimental permeability data yielded mean capillary wall pore diameter estimates ranging from 13–17 nm for the nasal respiratory vasculature compared to <10 nm for the vasculature in olfactory regions. For context, the apparent hydrodynamic diameter of IgG is ~ 10 nm. Thus, intranasal targeting of drugs to the olfactory regions may favor brain delivery due in part to reduced clearance into the systemic circulation.
Wednesday, May 9th, 2018
Room 2006, Rennebohm Hall
School of Pharmacy: 777 Highland avenue