ReviewAgile delivery of protein therapeutics to CNS
Graphical abstract
Brain delivery of proteins has been improved through several chemical modification strategies including cationization, fusing with cell-penetrating peptides (CPPs), fatty acylation, and conjugation with brain targeting ligands or block copolymers.
Introduction
Protein therapeutics has made significant progress during the past 30 years, beginning with the invention of the first recombinant protein used in clinical practice, a human insulin [1]. Since then, development of protein therapeutics has been one of the biotech's most notable successes. In recent years, the number of protein-based therapeutics reaching the marketplace has increased exponentially. As of today, more than 130 proteins or peptides are used in clinics and many more are in development [2]. The currently marketed proteins include enzymes, antibodies, clotting factors, anticoagulants, modern insulins, growth hormone, follicle-stimulating hormone, hematopoietic growth factors, interferons, interleukins and others. The market of the therapeutic proteins holds tremendous potential for future growth and it is estimated that by the end of 2018, it may reach the mark of US $165 billion as new products may enter the sector. As patents on first-generation proteins wind down, the industry seeks to protect their markets by introducing protein delivery technologies that provide for improved stability, bioavailability and safety of the therapeutic proteins. Such technologies aim to overcome obstacles to the clinical application of the proteins due to a lack of desirable attributes for adequate absorption or distribution. It therefore becomes critical to incorporate proteins in safe, stable and efficacious delivery systems. Because proteins face formidable enzymatic and penetration barriers, efficient protein delivery to its destination in the body remains a very challenging if not a formidable task.
There is a tremendous potential to develop protein therapeutics for the treatment of neurological and neurodegenerative disorders. Examples include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), human immunodeficiency virus 1 (HIV-1)-associated dementia (HAD) (or more generally HIV-associated cognitive dysfunction), multiple sclerosis (MS), lysosomal storage disorders (LSDs; Gaucher's disease, Niemann–Pick disease, Tay–Sachs disease and Sandhoff's disease, Krabbe's disease, Fabry's disease, metachromatic leukodystrophy amongst nearly 50 total disorders) and others. Other diseases associated with the central nervous system (CNS) include brain tumors, stroke, traumatic brain injury (TBI), and metabolic disorders. Some examples of potential protein therapeutics to treat these CNS related disorders include enzymes in LSDs, antibodies in AD and brain tumors, neurotrophic factors in PD and stroke, and gut–brain hormones in obesity.
Clinical use of these proteins, however, is extremely challenging because of the unique and complex environment imposed by the CNS. Systemic delivery of proteins to the brain inevitably encounters two major hurdles: the rapid serum clearance and the limited penetration at the blood–brain barrier (BBB). Some protein molecules, such as neurotrophic factors can cross the BBB to some extent but are rapidly cleared from the blood, whereas others, such as antibodies, are stable and long circulating in blood but absolutely not permeable at the BBB. In both cases systemic delivery of proteins does not allow to attain their sufficient brain concentration for effective treatment. Proteins can also access the brain through alternative delivery routes that allow bypassing the BBB, such as intracerebroventricular (i.c.v.), intraparenchymal, intranasal (i.n.) or intrathecal (i.t.) administration. However, in most cases the brain uptake of proteins following such administration routes is still surprisingly low, especially in the targeted brain regions where protein therapeutics needs to be delivered. It has been gradually accepted that serious biological barriers are associated with each of these alternative delivery routes.
Therefore a great deal of effort has been dedicated to developing the drug delivery systems and approaches that could help protein molecules crossing numerous barriers on their way to the site of action in the brain. Multiple drug delivery strategies were explored in the attempts to address this challenge. For example, chemical modification of proteins with poly(ethylene glycol) (PEG), known as PEGylation [3], or incorporation of proteins into poly(d,l-lactic-co-glycolide) (PLGA) particles [4], [5] increased stability and bioavailability of certain proteins and resulted in development of the Food and Drug Administration (FDA) approved products for various peripheral diseases. However, neither of these technologies has shown much promise so far in delivering protein therapeutics to the brain for treatment of CNS related diseases. Several specific molecules (antibodies, peptides, etc.) that can target and cross BBB through intrinsic transport systems available in brain endothelium were identified and conjugated to protein of interest to create targeted therapeutic agents for CNS related diseases. However, no such conjugate has progressed far enough to enter clinical trials although similar conjugates with small molecule drugs seem to be somewhat more advanced (e.g. paclitaxel–Bp-2 ANG1005, Angichem, Inc.). Some of the studies in this area go back nearly 30 years, and yet during this considerable period, despite consistent and steady effort by numerous capable researchers across the globe relatively little progress was achieved, which only underscores the enormity of the task.
However, analysis of previous experience in this field along with understanding of the recent achievements and trends in the drug delivery and nanomedicine science allow us to suggest that a new explosive development is just behind the corner. We believe that investigators should expect a very exciting journey during the next decade in pursuit of novel CNS technologies and therapeutics and that a critical mass of knowledge has been reached enabling new principal breakthroughs. In anticipation of this development we decided to critically analyze the past experiences from the current prospective that in our view in essential to achieve success in this field. We believe that the recent dramatic improvement in understanding the molecular physiology of CNS environment and the various barriers that exist on the way of successful protein delivery to the brain will be conductive to future progress. There is growing realization that the BBB, as part of the neurovascular unit (NVU), represents an interactive, dynamic, regulatory interface between the CNS and peripheral tissues [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. It is also clear that various pathological processes associated with neurological and neurodegenerative disorders alter the NVU and cause BBB dysfunction, which brings some opportunities and challenges to the design of protein therapeutics for these disorders. The choice of the routes of administration of these therapeutics is also pivotal and requires consideration of the disease stage (chronic or acute), location within the brain (widespread or local), and chemical nature of the compound to be delivered. We also believe, that there is a great opportunity in using nanomedicine approaches to improve the site-specific delivery and brain regional distribution of proteins administered though non-conventional routes allowing to avoid the BBB. It should be noted that due to small amounts of substances that can enter the brain, robust and reliable bioanalytical assays are needed for the analysis of the pharmacokinetics (PK) and biodistribution of the protein therapeutics. Carefully designed PK studies and proper interpretation involving analysis of PK and pharmacodynamics correlations and dose–responses are absolutely essential. Development of animal models that closely recapitulate human diseases and understanding of the limitations of these models are needed to carefully interpret results of the preclinical animal studies and use these results as for guidance for clinical trials. Here we present the readers with this review which briefly and sequentially considers the 1) BBB physiology and pathology in CNS related disorders; 2) main classes of protein and peptide therapeutics for CNS; 3) delivery routes for protein therapeutics; 4) chemical modification of proteins for CNS delivery; and 5) particle-based carriers for CNS delivery of proteins. We hope to disseminate and advance an in-depth understanding of each of these strategies and provide useful information for future design of protein delivery to the brain.
Section snippets
BBB physiology and pathology in CNS related disorders
Discovery of BBB is usually ascribed to the work of Paul Ehrlich and Edwin Goldman over a hundred years ago. They observed that intravenously injected dye stained all the organs with the exception of the brain and that the same dye exclusively stained the brain after injection into the brain [16], [17]. Thomas Reese and Morris Karnowsky further demonstrated that the blood was separated from the brain at the level of brain microvessel endothelial cells (BMECs). Under high resolution electron
Main classes of protein and peptide therapeutics for CNS
Current efforts in development of CNS biotherapeutics have focused on several classes of molecules including gut–brain hormones, lysosomal enzymes, neurotrophic factors, antibodies, and peptides. Some of the proteins and peptides evaluated for various neurological disorder indications in patients or approved for clinical use are listed in Table 1. All these molecules are believed to act upon targets in the CNS, which underscores the importance of their delivery to the brain. One class is
Delivery routes for protein therapeutics
The path of a therapeutic agent to its target organ and tissue begins at the site where the molecule is given to the body. Most current protein therapeutics including FDA approved products (e.g., antibodies and hormones) are administered by parenteral injection into fat tissue (subcutaneous, (s.c.)), muscles (intramuscular, (i.m.)) or veins (intravenous, (i.v.)). In few cases enteral and pulmonary routes were also explored to deliver protein therapeutics that requires frequent dosing to attain
Chemical modification of proteins for CNS delivery
To date some of the most extensive studies to increase protein permeability at the BBB have involved protein chemical modification with various strategies such as a) cationization, b) fusion with cell-penetrating peptides (CPPs), c) fatty acid acylation, d) conjugation with brain targeting ligands, and e) modification with polymers (Fig. 3). Notably, the protein modification points, linkers, modification degree and the conjugation chemistry are all important design considerations having a
Particle-based carriers for CNS delivery of proteins
Numerous studies have shown that encapsulation of therapeutic proteins in nano- or micron size particles decreases protein immunogenicity and improves protein stability and circulation time (Fig. 4). Liposomes and PLGA nanoparticles are possibly the most extensively investigated types of carriers for protein delivery. Other systems investigated in the context of CNS delivery include poly(butylcyanoacrylate) (PBCA) nanoparticles, and more recently, polyion complexes. Some other materials such as
Conclusion
Developing protein therapeutics for treatment of CNS disorders is an unmet need. A variety of delivery strategies discussed in this review have shown promise to delivery proteins to the brain. The most advanced in clinic are the strategies involving direct delivery of proteins to the CNS using the central administration routes, i.c.v. and intraparenchymal, as well as i.t. administration. Recently intranasal administration in the vicinity of nasal cribriform plate, which allows substances to
Acknowledgment
We would like to acknowledge the support of the National Institutes of Health RO1 NS051334, the Center of Biomedical Research Excellence (CoBRE) Nebraska Center for Nanomedicine P20 GM103480 (P20 RR021937) and the Russian Ministry of Science and Education Megagrant award (Contract 11.G34.31.0004) as well as the Carolina Partnership, a strategic partnership between the UNC Eshelman School of Pharmacy and the University Cancer Research Fund through the Lineberger Comprehensive Cancer Center. We
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