OverviewWe like to think the brain is special. In fact, all the neurons and associated satellite cells throughout the mammalian central nervous system (CNS)—which includes the spinal cord—comprise a highly specialized tissue. The complex circuitry executed by the CNS requires a unique cellular environment. The circulatory system maintains the fluid bathing most bodily tissues, but the CNS inhabits its own cerebrospinal fluid (CSF). In addition, a poorly understood system of cell–cell connections defines a "blood–brain barrier" (BBB) that segregates the brain from the blood vasculature to maintain distinct local chemical conditions. BBB integrity is crucial for normal health, but it also presents a challenge to drug delivery. Although a variety of hereditary and age-related cognitive disorders, as well as certain brain cancers, may prove responsive to drug treatment, any potentially therapeutic chemical must bypass the BBB first.
The October 25, 2011, meeting of the Biochemical Pharmacology Discussion Group addressed Brain Barriers: A Hurdle for Drug Discovery and brought together biochemists, clinical pharmacologists, and industry specialists to explore current BBB research. Speakers considered the physiological nature of the BBB, emerging techniques to transiently disrupt the BBB or to allow drugs to circumvent it, and the risks associated with these techniques.
In her introductory remarks Jo Ann Dumin, an organizer of the Brain Barrier event and researcher at Satori Pharmaceuticals, highlighted the intersector nature of the field by calling for a non-competitive consortium between academia and industry to solve the BBB challenge. The nascent research presented throughout the day and the intersection of ideas born from multiple disciplines underscored her suggestion.
Joel Pachter of the University of Connecticut Health Center began by demonstrating that physiologists do not agree which level of vascular organization or subcellular structures designate the BBB. Definitions range from general, citing only endothelial cells lining blood vessels, to detailed, naming specific tight junctions that lash cells together. Pachter argued that rigorous biological knowledge of BBB construction must inform clinical work. This notion drives research in his laboratory where experimental systems have been devised to identify relevant components of the BBB. He showed that advanced fluorescent microscopy can be used to calculate the densities of tight junctions on different blood vessel surfaces. Capillaries top the list in this assay for tight junctions per unit surface area, suggesting the vessels are key to the BBB.
Pachter further explained that cultured endothelial cells derived from brain tissue and used routinely in laboratories may not accurately reflect the BBB, because the vascular population from which they originated cannot be readily defined. To get a better understanding of which vessel population(s) contribute to the BBB, he proposed using laser capture microdissection, which allows for selection of specific cells from a heterogeneous population. The technique can be used to analyze endothelia from distinct types of vessels separately, allowing for comparisons of global gene and protein expression patterns. Pachter concluded by emphasizing the perhaps underappreciated heterogeneity of endothelial tissues.
Echoing Joel Pachter's introduction, Damir Janigro of the Cleveland Clinic pointed out that even apparently trivial questions about the BBB remain unanswered. His talk pivoted around the effects of induced, and even pathological, BBB disruption. Janigro's team studies epilepsy and the associated seizures, among other neuropathological disorders. Their analyses of a rat model of epilepsy in which pilocarpine administration induces seizures revealed that the drug does not significantly penetrate the BBB under any conditions, but it does precipitate BBB disruption. Direct injection into the brain resulted in loss of consciousness without convulsions. These observations suggested that mere BBB breach may cause seizures. A battery of experiments in model organisms correlated BBB opening with seizure onset, as tracked by electroencephalography (EEG), a measure of electrical activity in the brain.
Janigro's group also tested the hypothesis in human patients undergoing clinical surgeries and found that elevated serum S100β—a molecular marker of a breached BBB—predicted seizure onset. These experiments informed clinical work by Janigro and his colleagues on certain forms of epilepsy, but the results are also pertinent to the development of intentional BBB disruption methodologies for the purposes of drug delivery. Janigro introduced the ex situ endothelial culture tubes used in his laboratory to screen for the effects of various drugs on BBB permeability. The plastic tubes are seeded with human endothelial cells and allowed to develop into "mock" blood vessels. By flowing buffer through the vessels (mimicking blood flow) the researchers can observe the effect of shear forces on endothelium, while the permeability of the cell layer serves as a bench-top model of the BBB.
The day's lectures centered on the endothelial barrier between blood and brain tissue, but Adam Chodobski from the Warren Alpert Medical School at Brown University spoke about the less appreciated blood–cerebrospinal fluid barrier (BCSFB). The BCSFB resides in the choroid plexus, a highly vascularized organ consisting of epithelial cells, which is located within the ventricles (CSF-filled cavities) of the brain and is responsible for generating CSF. Since this liquid bathes the brain and spinal cord, it too is segregated from blood. The separation is similar to but distinct from that characterizing the BBB, and therefore represents an additional facet of brain-targeted drug delivery. Chodobski pointed out that CSF, which turns over four times a day in an adult human and drains into the peripheral circulation, plays an important role in clearance of drugs from their target sites. As delivery routes, the BCSFB and CSF may be useful for targeting brain cells abutting the CSF space, but other brain regions may be too far for effective distribution of introduced chemicals.
David Begley of King's College London began by reiterating two themes that cut across all the talks. First, the endothelium of the BBB is not homogeneous; general descriptions of its molecular properties therefore fail to provide a complete picture. Second, CNS vasculature is distinct from peripheral vasculature. Specifically, peripheral capillary cells are fenestrated: not sealed together by tight junctions, which allows solutes, large molecules, and even cells to pass between them. Peripheral capillary cells also routinely transport certain molecules by engulfing them on one surface, and releasing them on the opposite surface. Current dogma holds that the BBB does not transcytose in this way, but Begley's research demonstrates otherwise. He and his collaborators observed that although receptor-mediated transcytosis across the BBB occurs relatively rarely, it nevertheless contributes non-negligibly to BBB permeability. The range of BBB-active receptors is poorly characterized, but at least some are known and appear to exhibit limited but still useful flexibility in the classes of molecules they recognize. This opened the door to a novel delivery method: nanoparticles. The engineered micron-sized beads are coated with a marker that docks to receptors mediating endocytosis. Initial trials have demonstrated that intravenously injected nanoparticles pass through the rodent BBB and end up in neuronal cytoplasm within 30 minutes. The beads degrade in the cell with time. Further pharmacological and materials science research will address precisely how the nanoparticles can serve as a platform for specific drugs.
A cell within the rat brainstem takes up a nanoparticle by receptor-mediated endocytosis. In his keynote presentation David Begley of King's College London highlighted endocytosis as a historically underappreciated route to bypassing the blood–brain barrier. (Image courtesy Anja Zensi, University of Frankfurt and presented by David Begley) Innovative technology also featured in the presentation by Elisa Konofagou. Her group at Columbia University experiments with focused ultrasound (FUS) as a noninvasive tool to transiently open the BBB. FUS targets brain regions selectively by disrupting only a small (mm2- to cm2-sized) defined area of the BBB, with most of the disrupting effects localized to the acoustic focus. As a high-frequency sound emitted by FUS propagates across living tissue, it imparts mechanical perturbations, which in turn generate heat. The secondary thermal effect can precipitate cell damage, but this can be minimized by an injection of microbubbles (already established and approved as safe in other applications). The microbubbles resonate with the applied FUS frequency and cause BBB opening without generating excessive heat.
MRI imaging of murine and primate brains during FUS revealed the size and duration of "passive cavitations" under various conditions, and the laboratory has honed in on a set of acoustic frequencies and microbubble sizes that generate cavities of desired area and duration. Because FUS physically breaches the BBB, it can be applied broadly to any drug that can be delivered intravenously. A graduate student in the Konofagou laboratory, Yao-Sheng Tung, presented his work on identifying the ideal microbubble size. Tung's poster received one of two prizes awarded in the symposium's poster competition.
Our molecular understanding of some CNS-associated disorders suggests that gene therapy, rather than drugs, may prove therapeutic. Gene therapy involves the targeted delivery of genetic material to cells otherwise lacking the correct genetic information. Getting genes into cells is challenging enough, but getting them past the BBB poses an even greater hurdle. Brian Kaspar of the Ohio State University and The Research Institute at Nationwide Children's Hospital studies the pathology and therapeutics of neuromuscular disorders, which often stem from problems behind the BBB. Kaspar and his colleagues have found that adeno-associated virus 9 (AAV9) can bypass the BBB. Since viruses are essentially small capsules capable of delivering genetic payloads to very specific targets, this observation opens many avenues for future research. Preliminary studies have demonstrated the feasibility and tolerability of the approach in non-human primates.
Conor P. Foley, one of two winners of the poster competition, described arterial delivery of gene therapy in mice: injection of a viral vector into a specific artery feeding only a target region of the brain, coupled with chemically-induced BBB disruption delivered high doses of the vector without the need for surgery.
William H. Frey II of the Health Partners Alzheimer's Research Center at Regions Hospital and the University of Minnesota spoke of avoiding the BBB altogether when targeting drugs to the CNS. Extracellular pathways connect the nasal passage essentially directly to the brain cavity in the region where olfactory nerves communicate between the two compartments. Intranasal delivery is therefore noninvasive, amenable to a wide range of chemical compounds, and functional with low drug doses. An interest in low glucose uptake by brain tissue in Alzheimer's patients led Frey and his colleagues to successfully demonstrate the effectiveness of intranasally administered insulin. More recently, intranasally administered stem cells in a rat model of neurodegenerative disease were shown to migrate into the CNS, with manifest possibilities for future clinical research.
