The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance, as well as several ongoing intrathecal clinical trials. In the present study, we clarify how IgG and smaller single-domain antibodies (sdAb) distribute throughout the whole brain in a size-dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo three-dimensional magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along the perivascular spaces of all vessel types, with sdAb accessing a four- to seven-fold greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size-dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points allowing substances in the CSF to enter the perivascular space. Osmolyte co-infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by ∼50%. The results of the present study reveal potential distribution mechanisms for endogenous IgG, which is one of the most abundant proteins in the CSF, as well as provide new insights with respect to understanding and improving the drug delivery of macromolecules to the central nervous system via the intrathecal route.
The intranasal route has been hypothesized to circumvent the blood-brain and blood-cerebrospinal fluid barriers, allowing entry into the brain via extracellular pathways along olfactory and trigeminal nerves and the perivascular spaces (PVS) of cerebral blood vessels. We investigated the potential of the intranasal route to non-invasively deliver antibodies to the brain 30 min following administration by characterizing distribution, dose-response, and mechanisms of antibody transport to and within the brain after administering non-targeted radiolabeled or fluorescently-labeled full length immunoglobulin G (IgG) to normal adult female rats. Intranasal [I]-IgG consistently yielded highest concentrations in the olfactory bulbs, trigeminal nerves, and leptomeningeal blood vessels with their associated PVS. Intranasal delivery also resulted in significantly higher [I]-IgG concentrations in the CNS than systemic (intra-arterial) delivery for doses producing similar endpoint blood concentrations. Importantly, CNS targeting significantly increased with increasing dose only with intranasal administration, yielding brain concentrations that ranged from the low-to-mid picomolar range with tracer dosing (50 μg) up to the low nanomolar range at higher doses (1 mg and 2.5 mg). Finally, intranasal pre-treatment with a previously identified nasal permeation enhancer, matrix metalloproteinase-9, significantly improved intranasal [I]-IgG delivery to multiple brain regions and further allowed us to elucidate IgG transport pathways extending from the nasal epithelia into the brain using fluorescence microscopy. The results show that it may be feasible to achieve therapeutic levels of IgG in the CNS, particularly at higher intranasal doses, and clarify the likely cranial nerve and perivascular distribution pathways taken by antibodies to reach the brain from the nasal mucosae.
Intranasal administration provides a non-invasive drug delivery route that has been proposed to target macromolecules either to the brain via direct extracellular cranial nerve-associated pathways or to the periphery via absorption into the systemic circulation. Delivering drugs to nasal regions that have lower vascular density and/or permeability may allow more drug to access the extracellular cranial nerve-associated pathways and therefore favor delivery to the brain. However, relative vascular permeabilities of the different nasal mucosal sites have not yet been reported. Here, we determined that the relative capillary permeability to hydrophilic macromolecule tracers is significantly greater in nasal respiratory regions than in olfactory regions. Mean capillary density in the nasal mucosa was also approximately 5-fold higher in nasal respiratory regions than in olfactory regions. Applying capillary pore theory and normalization to our permeability data yielded mean pore diameter estimates ranging from 13–17 nm for the nasal respiratory vasculature compared to <10 nm for the vasculature in olfactory regions. The results suggest lymphatic drainage for CNS immune responses may be favored in olfactory regions due to relatively lower clearance to the bloodstream. Lower blood clearance may also provide a reason to target the olfactory area for drug delivery to the brain.
Passive immunotherapy, i.e., the administration of exogenous antibodies that recognize a specific target antigen, has gained significant momentum as a potential treatment strategy for several central nervous system (CNS) disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and brain cancer, among others. Advances in antibody engineering to create therapeutic antibody fragments or antibody conjugates have introduced new strategies that may also be applied to treat CNS disorders. 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 and blood-cerebrospinal fluid barriers that greatly restrict transport of peripherally administered molecules from the systemic circulation into the CNS. Here, we summarize the various passive immunotherapy approaches under study for the treatment of CNS disorders, with a primary focus on disease-specific and target site-specific challenges to drug delivery and new, cutting edge methods.
The parathyroid hormone receptor-1 (PTHR1) is a member of the B-family of GPCRs; these receptors are activated by long polypeptide hormones and constitute targets of drug development efforts. Parathyroid hormone (PTH; 84 residues) and PTH-related protein (PTHrP; 141 residues) are natural agonists of PTHR1, and an N-terminal fragment of PTH, PTH(1−34), is used clinically to treat osteoporosis. Conventional peptides in the 20-40-mer length range are rapidly degraded by proteases, which may limit their biomedical utility. We have used the PTHR1-ligand system to explore the impact of broadly distributed replacement of α-amino acid residues with β-amino acid residues on susceptibility to proteolysis and agonist activity. This effort led us to identify new PTHR1 agonists that contain α→β replacements throughout their sequences, manifest potent agonist activity in cellular assays, and display remarkable resistance to proteolysis. The strategy we have employed suggests a path toward identifying protease-resistant agonists of other B-family GPCRs.
Passive immunotherapy has become a prominent therapeutic strategy for the treatment of many different neurological disorders. Antibodies can be targeted to the pathogenic proteins of diseases such as Alzheimer's (e.g., beta‐amyloid and phosphorylated tau), Parkinson's (e.g., alpha‐synuclein), and multiple sclerosis (e.g., CD20 marker on B cells). However, delivery of antibodies to the central nervous system (CNS) following systemic administration is significantly impeded as a result of the blood‐brain barrier (BBB) and blood‐cerebrospinal‐fluid barriers (BCSFBs). The intranasal route has been hypothesized to circumvent the BBB and BCSFBs and allow entry into the brain via extracellular pathways along cranial nerves and the perivascular spaces (PVS) of cerebral blood vessels (Thorne et al. Neuroscience 2004 & 2008; Lochhead et al. J. Cereb. Blood Flow & Metab. 2015). Here, we demonstrate the potential of intranasal delivery as a non‐invasive strategy to bypass the BBB and BCSFBs to rapidly target full length immunoglobulin G (IgG) antibodies to the CNS. Quantitative distribution studies of radiolabeled IgG in the CNS of anesthetized rats revealed that intranasal delivery achieved significantly higher antibody levels in both the CNS and CSF compared to systemic delivery at doses resulting in matched blood exposure via either route. Additional experiments exploring the intactness of radiolabeled IgG in the brain following intranasal delivery were conducted to identify the biochemical conditions necessary for IgG intactness in vivo. Finally, by utilizing fluorophore‐labeled IgG, we provide direct evidence of intranasal IgG transport along perineural and perivascular pathways that directly connect the nasal mucosa to CNS target sites. Our results suggest olfactory and trigeminal nerve‐associated perineural channels provide direct pathways to the olfactory bulbs and brainstem, respectively. Once within the brain, further distribution along perivascular spaces of cerebral blood vessels is capable of transporting IgG to widespread regions of the brain. Our findings highlight that the intranasal route can provide a non‐invasive, direct, and rapid method for targeting antibodies to the CNS, bypassing the BBB and BCSFBs in a way that merits further consideration for the treatment of neurological diseases.Support or Funding InformationSupported by the University of Wisconsin‐Madison School of Pharmacy, the Graduate School at the University of Wisconsin, the Michael J. Fox Foundation for Parkinson's Research, the Parkinson's Foundation, the Wisconsin Alzheimer's Disease Research Center (NIH P50‐AG033514), the Wisconsin institute for Clinical and Translational Research (Clinical and Translational Science Award program through the NIH National Center for Advancing Translational Sciences, grant UL1TR000427), and the Wisconsin Alumni Research Foundation grant 135 PRJ82AZ.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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