Department of Anaesthesia and Critical Care, University of Würzburg, 97080 Würzburg, Germany
* Corresponding author Email: Foerster_C@ukw.de; E_Shityako_S@ukw.de
The existence of the blood–brain barrier in the human body leads to the insufficiency in delivering therapeutic compounds into the brain for the effective treatment of various neurological disorders. In order to determine the possibility of such agents to penetrate through the blood–brain barrier, different
We carried out a literature research and study selection by searching for published biomedical articles in the PubMed archive.
Overall, the combination of
In the drug discovery process, drug permeation across the blood–brain barrier (BBB) is fundamental for neuropharmaceuticals to reach their site of action within the central nervous system (CNS). This BBB consists of highly specialised microvascular endothelial cells together with pericytes, astrocytes, microglia, neurons and basement membrane. The capillary endothelial cells are connected by proteins (occludin, claudins and junctional adhesion molecules) forming tight junctions (TJs), which seal the intercellular space, thereby restricting the permeability for the CNS-active substances[2,3]. In addition, these cells contain numerous active membrane transporters to regulate transcellular transport of drug-like molecules and their metabolites between the blood–brain interface.
Over 98% of all known therapeutics are unable to penetrate the BBB due to their molecular properties and physicochemical factors, including hydrophilicity, hydrophobicity, polar surface area, molecular size and charge (Figure 1). On the contrary, the permeation of the CNS-inactive compounds would generate various undesired side effects. Receptor-mediated and non-specific adsorption-mediated transcytosis can also contribute to the translocation of peptides, antibodies and lipoproteins across the BBB. To minimise this risk, the healthy BBB itself imposes a highly efficient impediment for most of the clinically administered neuropharmaceuticals. On the other hand, the BBB dysfunction is highly implicated in auto-immune, neuropathological processes (Alzheimer and Parkinson’s diseases), neuroinfections (meningitis and encephalitis), haemorrhagic and ischemic stroke and traumatic brain injury[5,6,7]. In this regard, the assessment of the BBB permeation for drug candidates at physiological and pathological conditions would be a primary concern for rational drug design and development through various
Schematic depiction of the blood–brain barrier permeability for different drug-like chemical substances.
The goal of this review is to describe the state-of-the-art techniques and methods that have been used so far in pharmaceutical research to evaluate the BBB function and assess the ability of drug-like molecules to permeate the BBB.
We performed a literature search and study selection by seeking published biomedical research papers in PubMed. The criteria for search were as follows:
• article type: review, research article
• publication date: various
• species: mammals
• language: English
• key words: blood–brain barrier,
We also used monographs dedicated to the BBB research and drug design strategies to bring readers the state-of the-art information in regard to describing issues.
The authors have referenced some of their own studies in this review. These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. All human subjects, in these referenced studies, gave informed consent to participate in these studies.
To screen the virtual libraries that encompass up to hundreds of thousands and even millions of drug-like molecules, numerous procedures were devised based on their molecular descriptors and fingerprints. A standard high-throughput screening (HTS) is a method of choice to filter and determine the CNS-active drug/hit/lead-like compounds either by the descriptor- or by the molecular docking-based strategy (Figure 2).
Schematic depiction of
As for the descriptor-based HTS, the great assessment in this direction was done by Lipinski and co-authors, characterised in a literature as the Rule of Five. Despite the fact that the Rule of Five was widely adopted by both pharmaceutical industry and academia for its robustness (few false-negatives) and fast calculation speed, there were disadvantages of the method. Among those were lots of false-positive outcomes due to simple summation of molecular properties (molecular weight, sum of nitrogen and oxygen atoms, etc.) without considering the BBB transport mechanisms, such as multidrug P-glycoprotein (P-gp) transporter efflux and strong reliance on experimentally determined datasets.
Aside from the Lipinski’s rule of thumb, the other methods were also elaborated to predict the ability of substances to permeate the BBB successfully and exert their pharmacological potential. Among them are various quantitative structure-activity relationship (QSAR) regression models based on the BBB partitioning values, such as logBB, taken from experimental data for various drug-like molecules. The logBB parameter is defined as the logarithm value of steady-state brain to blood (plasma) concentration ratio for a drug of interest according to following equation:
In molecular descriptor-based analysis, the predicted logBB parameter was mainly derived from the notion of molecular polar surface area descriptor and octanol-water partition coefficient (logP) to assess compound hydrophobicity and H-bonding capacity (desolvation rate). These two last descriptors were vigorously discussed throughout the literature[10,11]. For instance, they were implemented continuously in the QSAR regression models through many mathematical formulas, such as Clark and Rishton equations[12,13]:
On the other hand, the molecular docking-based methods have been successfully used to determine the P-glycoprotein substrates or inhibitors dealing with the phenomenon of active multidrug efflux by P-gp in the brain[14,15]. Despite its relative precision, this approach depends on the accurate crystallographic three-dimensional models of the protein structure and implements laborious ligand-receptor preparations and computationally slow genetic algorithms[16,17]. Therefore, this approach is particularly valuable when used in combination with previously described methods to exclude the role of active transport as a result of drug-P-gp interaction upon the BBB permeation of drug-like chemical compounds.
In CNS research, BBB permeability properties of drug-like candidates are very important. Systems that can be used for HTS are favoured. Moreover, methods that allow for direct access to the brain endothelium with no interference from other brain structures are preferred.
Cells of both cerebral and non-cerebral origin are used as
Isolated brain capillaries are used for BBB transport studies[23,24,25]. Freshly isolated capillaries directly reflect the situation at the luminal side of brain capillaries. In fact, they reflect the
Primary BCECs also mimic the
In order to overcome the problems concerning reproducibility of primary BCECs, immortalised BCECs are established for
Improvement of barrier properties has also been reported in porcine cerebral capillary endothelial cells, rat BCECs and human dermal microvascular endothelial cell line (hDMEC/D3). Meanwhile, BCECs can also be co-cultured with astrocytes, C6 glioma cells or pericytes to improve barrier properties. These cells can be grown either with or without contact with BCECs in a Transwell culture system. Transwell models have been developed to study BBB permeation. Most permeability experiments employ this method. It can be a monodimensional system, wherein which only BCECs are grown on a microporous membrane or a two-dimensional system wherein the BCECs are co-cultured with other cells (Figure 3A–C).
Schematic drawing of static
However, all these systems lack the experimental replication of intraluminal blood cells together with bloodstream flow that imparts shear stress as it is occurring
To compensate for the aforementioned lack in shear stress as affecting endothelial barrier function, dynamic BBB models were established. In these models, hollow fibres that mimic capillaries and allow co-culture of other cell types were used (Figure 4). Bovine aortic endothelial cells co-cultured with glial cells were the first BBB model to adopt this method. More recently, immortalised porcine brain endothelial cells co-cultured with glial cells were used. The human cerebral microvascular endothelial cell line (hCMEC/D3) co-cultured with astrocytes grown in the lumen of hollow microporous fibres and exposed to a physiological pulsatile flow was also recently developed as a dynamic BBB model. This method demonstrated that hCMEC/D3 cells cultured under pulsatile flow conditions have maintained
Schematic representation of a conventional dynamic
There are several
There are various
Schematic diagram of different invasive
The HPLC analysis of mouse or rat brain homogenates is a crude method of choice; it starts with a homogenate preparation by ultrasonication in Dulbecco’s phosphate-buffered saline (PBS) or other matrices following high-speed centrifugation to produce a clear supernatant for further determination of drug concentration by HPLC. The major disadvantage of this approach is that the residual capillary blood in the brain might influence the results and, therefore, should be eliminated via brain reperfusion with PBS before surgery or
After perfusion, the animals are decapitated and the brain is analysed for reference and test compounds to quantify the logPS coefficient, which is a calculation method based on the rate of brain penetration for analysed chemical entity. The logPS parameter is calculated as follows:
Intracerebral microdialysis is a valuable tool in pharmaceutical research that is used to perform a direct sampling of cerebral interstitial fluid via establishing a dialysis catheter with semipermeable membrane into the brain. Therefore, the molecule of interest from the brain will traverse this membrane according to its concentration gradient (from high to low) that makes it possible to analyse within the collected fluid (microdialysate)[49,50]. This technique allows the possibility to monitor the drug concentration in the brain over time within the same animal and probe different brain areas as presumable drug targets. The potential drawbacks may include chronic BBB inflammation and disruption caused by this invasive procedure followed by increased BBB leakage and plasma protein extravasation. Despite these shortcomings, this method of choice is still the only technique that provides information about the local concentration of unbound fraction of drug-like substances at any given time in freely moving animals.
Special thanks are extended to Anna Poon from the City College of New York for her assistance in the paper’s writing. The authors are grateful to the BMBF (Bundesministerium für Bildung und Forschung) for the support of this work by providing the grants (13NM803) to Carola Förster.
BBB, blood–brain barrier; BCEC, brain capillary endothelial cell; cEND, cerebrovascular endothelial cell line; CNS, central nervous system; HPLC, high-performance liquid chromatography; HTS, high-throughput screening; PBS, phosphate-buffered saline; QSAR, quantitative structure-activity relationship; TJ, tight junction.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.