The workshop focused on strategies for delivering drugs to the brain by crossing or bypassing the blood–brain barrier (BBB). Many of the approaches currently in development target neurodegenerative diseases, such as Alzheimer’s disease (AD). However, the pursuit of treatments for orphan indications, such as mucopolysaccharidosis types 1 and 2, may be more tenable as they may benefit from the Food and Drug Administration’s (FDA’s) Accelerated Approval Pathway, suggested Francesca Bosetti, program director for Stroke in the Neural Environment Cluster at the National Institute of Neurological Diseases and Stroke. In addition, individuals affected by rare serious diseases may be more motivated to participate in clinical trials. Hyman added that techniques for traversing or circumventing the BBB may also be applied effectively to psychiatric disorders, such as mood disorders and schizophrenia. Since these conditions could require speedier delivery of drugs to the brain as well as shorter-term persistence of the drug in the brain, different technologies and approaches may be needed, he said.
The BBB is a continuous endothelial membrane that, along with pericytes and other components of the neurovascular unit, limits the entry of toxins, pathogens, and blood cells to the brain, said Berislav Zlokovic, director of the Zilkha Neurogenetic Institute at the Keck School of Medicine, University of Southern California (Zlokovic, 2011). It accomplishes this through the actions of numerous transport systems. However, it also represents an obstacle to central nervous system (CNS) drug delivery, said
Zlokovic. Moreover, he said, the fact that blood vessel patterns tightly follow brain circuits suggests that the vascular system, and therefore the BBB, plays an important role in normal brain function, aging, and disease.
Breakdown of the BBB is associated with several neurodegenerative disorders, such as AD, amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), multiple sclerosis, and chronic traumatic encephalopathy, as well as stroke and infectious diseases of the brain, such as those caused by the human immunodeficiency virus, said Zlokovic. He added that there are also rare monogenic neurological diseases associated with genetic defects in nonneuronal cells that cause disruption of the neurovascular unit and breakdown of the BBB (Zhao et al., 2015). Some of these diseases are associated with a change in expression of transporters. For example, Zlokovic and colleagues have shown that reduced expression of glucose transporter 1 (GLUT1), which mediates glucose transport into the brain, is associated with neuronal dysfunction and degeneration in AD (Winkler et al., 2015). The question is whether these disease-associated gene alterations that are associated with BBB breakdown contribute to the disease or are innocent bystanders, said Zlokovic. While the workshop focused on mechanisms to enable delivery of drugs to the brain by crossing or bypassing the BBB, Zlokovic noted that a pathologically disrupted BBB is unhealthy, and that healthy blood vessels will be needed to deliver drugs to the brain. His laboratory has thus explored approaches to prevent disruptions of the BBB that may contribute to disease. In AD, for example, the pathogenic protein amyloid-beta (Aβ) interacts with a receptor for advanced glycation end products (RAGE), disrupting the BBB and enabling the transport of Aβ across the BBB, said Zlokovic. RAGE blockers are currently being tested in phase III trials to see if they can suppress the accumulation of Aβ in the brain (Deane et al., 2003). Zlokovic and colleagues are also investigating the mechanisms by which an enzyme called activated protein C (APC) may protect neurons by tightening the BBB (Griffin et al., 2015). A variant of APC called 3K3A-APC, which optimizes the cell-signaling properties of the enzyme (Griffin et al., 2016), is currently in phase II trials for stroke, said Zlokovic.
There have been many attempts over the past 25 years to develop biologics for brain diseases, said William Pardridge, professor of medicine at the University of California, Los Angeles. These include attempts to deliver neurotrophins to treat ALS and PD, and growth factors to treat stroke. The earliest efforts delivered the drugs by subcutaneous injection, not appreciating that they would not cross the BBB. In stroke, the BBB is
disrupted but not until 12 to 24 hours after the event, long past the “window of opportunity” for neuroprotection, said Pardridge. Subsequent efforts tried injections directly into the brain or through a technique called convection-enhanced diffusion. Although these studies showed that drug levels in the cerebrospinal fluid (CSF) exceeded those in the plasma, these trials were nevertheless unsuccessful, indicating that CSF levels do not reflect BBB penetration, said Pardridge. More recently, investigators have begun to exploit specialized transport systems in the microvasculature as a means of delivering drugs across the BBB, he said. These are the focus of this workshop.
During the workshop presentations and discussions, participants identified gaps and challenges associated with development of methods for traversing the BBB and to improve therapeutic delivery to the nervous system. The issues listed here, and attributed to the individuals who made them, are expanded on in succeeding chapters.
Understanding the Complex Biology of the BBB
The major reasons for the high attrition rates in CNS drugs include a limited understanding of drug permeability at the BBB, drug distribution in the brain, and target engagement in the brain, said Danica Stanimirovic. Understanding the BBB molecular makeup should enable identification of new targets for both managing disease and delivering drugs, she said. However, Robert Thorne, assistant professor in pharmaceutical sciences at the University of Wisconsin–Madison, called the study of the BBB an “orphan field,” which lacks the appreciation and funding it is due within the neuroscience community.
Zlokovic described the complex interplay of cells and molecules that shape BBB structure and function, including not only endothelial cells, pericytes, and vascular mural cells but also microglia and astrocytes, and molecules such as various BBB junction proteins, transporters, receptors, and ion channels (Zhao et al., 2015). Regional heterogeneity of these components adds further complexity to understanding structure and function, said Zlokovic.
The complex biology of the BBB contributes to the difficulty in studying many aspects of BBB disruption, including the role of circadian
rhythms and differences between wakefulness and sleep states, said Thorne, and the timing of its disruption during stroke, said Pardridge.
Understanding Drug Delivery and Distribution in the Nervous System
According to Thorne, attempts to deliver drugs systemically to the brain have not been fully translated to the clinic, in part because delivering drugs intended for the brain via systemic routes may result in unacceptably high levels in the periphery. Even direct infusions into the brain or CSF have had limited success, he said, in part because diffusion of large molecules, such as enzymes, from the CSF to the brain is limited. Other delivery approaches may be more promising, such as delivery to perivascular spaces—the fluid- and connective tissue–filled areas surrounding blood vessels in the subarachnoid space between the brain and the skull, said Thorne. However, achieving efficient drug delivery will require better characterization of the precise distribution of molecules in the CNS using different strategies, and a better understanding of how molecules move in the CSF and perivascular spaces, he said. In addition, he cited the need to examine other variables that may influence distribution of molecules in the CNS, including body position, intracranial pressure, effects of various diseases, and individual variations.
Viral delivery of drugs presents other challenges, said Viviana Gradinaru, Heritage principal investigator at the California Institute of Technology. Most work has been done with adenoassociated viruses, although these have a small packaging capability. There are also challenges with regard to region and cross-species specificity, she said. Also not well understood are the mechanisms underlying the use of focused ultrasound to facilitate drug delivery, said Alexandra Golby of Brigham and Women’s Hospital.
Creating Suitable Models for Understanding BBB Dysfunction
Zlokovic commented that available methods in humans to study BBB dysfunction do not distinguish among the different mechanisms of permeability, for instance paracellular via transcytosis versus transcellular via breakdown of tight junctions. While animal models can distinguish among these mechanisms, they present other challenges, he said. For example, anatomical and size differences between species affect both how a molecule crosses the BBB and whether it hits its desired target, said Douglas
Hunt, head of regulatory affairs for ArmaGen. Larger species also have more complex brains, said Stanimirovic. Differences in the availability of transporters and receptors, for example, can result in selective efficacy of biologics and antibodies, she said. Pharmacokinetic and pharmacodynamic studies may also not translate well between small and large animals, said Balu Chakravarthy, senior research officer at the National Research Council of Canada.
Despite the limitations of animal models, they are essential to develop potential biomarkers and to gather strong efficacy data before undertaking expensive preclinical studies and even more expensive clinical studies, said Bosetti.
Conducting Complex Preclinical Studies
The preclinical evaluation of complex molecules capable of traversing the BBB is not simple, but it is necessary to understand the attributes that would enable them to be moved through the development process into clinical translation, said Stanimirovic. Evaluating the toxicity of complex fusion molecules delivered to the brain presents multiple challenges, including immunogenicity, targeting of different brain areas, and the potential for cross-linking with surface proteins and evoking a cascade of high-level immune activation, known as a “cytokine storm,” said Matthew Whittaker, a pharmacology and toxicology reviewer at FDA.1 He added that even when drugs are designed to target the brain, systemic toxicity needs to be evaluated. Species-specific biologies add to the complexity not only in terms of toxicity but also in terms of efficacy, because different species may express different levels of a desired target receptor, said Hunt. He added that to evaluate dosing in preclinical studies, predictive models would be advantageous.
Addressing Barriers to Translation into Effective Treatments
Translational challenges include a lack of preclinical outcomes that mimic clinical outcomes and inadequate biomarkers, said Stanimirovic. Among existing biomarkers, CSF biomarkers that show a drug is getting into the brain may not correlate with improved clinical function, said Hunt. This raises ethical concerns about doing lumbar puncture in children, he
1 The discussion represents the views of the participants and should not be construed to represent FDA’s views or policies.
said. For orphan indications, key challenges include defining appropriate clinical end points and meeting enrollment goals, particularly in light of the heterogeneity of many of these conditions, said Hunt.
To accelerate translation, Stanimirovic also suggested a need to streamline regulatory pathways while managing toxicity, safety, and immunogenicity. Deepa Rao of FDA’s Division of Psychiatry Products noted that this can be particularly challenging when therapeutics combine multiple components, such as a drug and device, two enzymes, or an antibody and enzyme. Hunt added that determining the risk–benefit ratio for molecules that target the brain may be more complex than for drugs targeting other organ systems.
Streamlining Siloed Research Programs and Attracting Scientists to the Field
Progress in understanding the BBB and developing technologies to traverse it are also hindered by the manner in which research is conducted. For example, Frank Walsh, founder and chief executive officer of Ossianix, said that pharmaceutical companies operate in silos, such as pharmacology and toxicology, rather than integrating efforts toward a common goal. Pharmaceutical companies have also failed to appreciate the importance of delivery science, said Thorne.
Shortcomings in academia also contribute to slow progress in the BBB field, especially in its historical failure to attract and train a sufficient number of scientists to the field, said Thorne. Many academic neuroscience programs still overlook the cerebrovasculature and CNS barriers in their training even today, in part because they lack qualified faculty with appropriate expertise in CNS barriers science and vascular biology. Even if a pharmaceutical company wanted to resource a BBB program, it would find few trained scientists to hire, he said. Edmund Talley, program director of channels, synapses, and circuits at the National Institute of Neurological Diseases and Stroke, added that academia has underappreciated regulatory science.
Stanimirovic said that it is time to address the evolving concept of the BBB as a very dynamic fluid membrane that expresses many different transporters, shuttles, and pores; and that interacts very closely with other
elements of the environment, including glia, pericytes, neurons, and microglia. Only then can this new evolving knowledge of the BBB be exploited for developing new and more targeted drug-delivery approaches that combine novel molecules with innovative delivery strategies, she said.
Developing and Refining Novel Approaches for Drug Delivery
Innovation is now on the drug delivery side, said Pardridge. Proteins and polypeptides may now be delivered to the brain through a variety of approaches:
- Trojan horse approaches using fusion molecules (Walsh)
- Exploiting the inflammatory response by using macrophages and monocytes to transport therapeutic molecules (Kabanov)
- Using viral vectors to transport genes into the brain (Gradinaru)
- Disrupting the BBB with focused ultrasound to enable delivery of therapeutic molecules (Golby)
- Bypassing the BBB by delivering drugs to the brain intranasally, via the olfactory or trigeminal nerve pathways (Thorne)
Developing New Models That Better Represent the BBB
Translation of basic science to clinical applications is a complex process that will require increased development of both in vitro and animal models, said Stanimirovic.
- In vitro models have played a significant role in facilitating the discovery and development of new drugs for CNS diseases, said Choi-Fong Cho, a neuroscientist at Brigham and Women’s Hospital and Harvard Medical School. A spheroid model developed in her lab enables high-throughput drug screening.
- Stem cell technologies have enabled the development of 2-D and 3-D models to study BBB permeability and transport (Zlokovic).
- More than 40 animal models have been developed to study BBB breakdown in human disease and to tease out mechanisms that contribute to it, such as pericyte degeneration and fibrinogen deposition (Zlokovic).
- Systems biology approaches using mathematical modeling can help elucidate mechanisms and predict responses in terms of both safety and efficacy (Shah).
The field is increasingly moving toward the preclinical use of biomarkers of target engagement and toxicity, said Stanimirovic.
- Biomarkers could improve the efficiency of clinical trials, thus lowering their cost (Patel).
- Biomarkers that confirm drug delivery into the brain and BBB permeability status would enable subject stratification for clinical trials (Bosetti).
- Novel imaging and molecular techniques also provide opportunities to study brain vasculature and BBB disruption in living animals and humans, and correlate these with cognitive changes (Zlokovic).
Building Collaborations to Advance Understanding of the BBB
Because the BBB is complex and truly multidisciplinary, collaborative and cooperative research approaches are essential, said Stanimirovic. Bernd Stowasser, head of global public–private partnerships at Sanofi, and a core member of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Innovative Medicines Strategy Priority Workgroup, added that collaborations such as public–private partnerships provide benefits for all stakeholders: academic researchers, industry, and patients.
- Because the BBB occupies a unique interface between the vasculature and the brain, it may provide the opportunity for research dollars to have a substantial effect (Thorne).
- Advancing innovative BBB research will require an increased focus on basic and translational science, attracting more young researchers to the field, and supporting them to stay in the field (Brose, Gu, Lisanby, Talley, Thorne).
- Consortia could develop training programs and incentives to encourage scientists to enter and remain in the field (Thorne).
- Attracting the interest and expertise of investigators from outlying fields would encourage innovation in BBB science (Campany).