Despite the presence of paracellular TJs and AJs at the BBB, the glycocalyx, bulk flow and metabolic enzymes restricting the brain exposure to compounds, there are several routes by which essential nutrients and other molecules can be transported across the BBB (Fig. 2). In general, these routes include passive diffusion,
carrier-mediated transport and vesicular trafficking (see more detailes in chapter 2.3.1, 2.3.2, 2.3.3, respectively). These routes facilitate not only entry of endogenous compounds, but also can be used for drug delivery into the brain. In addition, immune cells such as macrophages and monocytes can be recruited to the brain in disease conditions by means of transcytosis (Davoust et al., 2008). Importantly, neuropathological conditions can affect the integrity and functioning of the BBB and alter transport of molecules.
Figure 2. Different routes of transport across the BBB. Passive diffusion and facilitated carrier-mediated transport across the lipid bilayer occur according to the concentration gradient in both directions from blood to brain or from brain to blood. ABC - ATP binding cassette transporters; AMT - adsorptive-mediated transcytosis; RMT - receptor-mediated transport;
SLC - Solute carrier transporters.
2.3.1 Passive diffusion
During long time, transcellular passive diffusion has been considered as the only way the compounds can cross the BBB and most of the brain delivery strategies were based on improvement of lipophilicity of the drugs. Passive diffusion is an energy-independent process occuring according to the concentration gradient of the unbound compounds on both sides of the cell membrane, which is directly proportional to the diffusion rate (Levin, 1980). The passive diffusion can be either paracellular (between the endothelial cells) or transcellular (through the endothelial cells)(Fig. 2). The diffusion between cells is considered negligible due to the presence of TJs between the endothelial cells. In contrast, highly lipophilic compounds with small molecular size such as heroin and diazepam can be transported across the BBB via transcellular diffusion.
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In terms of passive diffusion, there is a correlation between BBB permeability and physicochemical properties of the compounds with some exceptions (Table 1). A strong correlation between the drug lipophilicity and its BBB permeation rate was observed, where increase in lipophilicity resulted in higher permeability (Neuwelt et al., 2008). It can be explained by the fact, that increased lipophilicity of compounds leads to enhanced nonspecific brain tissue binding in the brain parenchyma and drives the transcellular diffusion according to the concentration gradient (Summerfield et al., 2007). However, one should remember, that only unbound drug can interact with the target within the brain. Therefore, higher brain nonspecific tissue binding and BBB permeation due to lipophilicity should be considered with caution. In addition, Waring (2009) investigated the relationship between permeability of structurally diverse compounds in human epithelial colorectal adenocarcinoma cells (Caco-2) and suggested the limits for the molecular weight and logD in order to achieve the BBB permeation (Table 1) (Waring, 2009). The drugs with high potential to form hydrogen bond decreases the permeation across the BBB (Pajouhesh and Lenz, 2005). Molecular topological polar surface area (TPSA) has been used to predict BBB penetration of drugs with the upper limit of 90 Å2 (Pajouhesh and Lenz, 2005; van de Waterbeemd et al., 1998). In general, weak basic and acidic compounds in uncharged form can pass across the membrane, therefore the negative logarithm of the acid dissociation constant, pKa, of the drug at the physiological pH 7.4 will also show effect on the BBB permeation (Fischer et al., 1998).
Table 1. Physicochemical properties associated with the BBB permeation of drugs.
Parameter Limit values Reference
Molecular weight vs. logD
<300 >0.5 300–350 >1.1 350–400 >1.7 400–450 >3.1
Waring, 2009
TPSA < 90 Å2 van de Waterbeemd et
al.,1998
H-bond donor atoms < 3 Pajouhesh and Lenz,
2005
H-bond acceptor atoms < 7 Pajouhesh and Lenz,
2005
pKa 4-10 Fischer et al., 1998
2.3.2 Carrier-mediated transport
The carrier-mediated transport (CMT) occurs after the interaction between transport protein expressed in the membrane of the cerebral endothelial cells and endogenous compounds or xenobiotics, which can be both lipid soluble or hydrophilic molecules.
The transporters can be expressed in either luminal or abluminal membrane or both sides of the endothelial cells (Table 2). Depending on transporter expression and function, the carrier-mediated passage of molecules (Fig. 2) can be unidirectional and denote either influx or efflux across the BBB or bidirectional meaning the transport of solutes in both directions across the BBB according to the concentration gradient (Begley, 2004). In addition, the CMT can be active or facilitated (also known as equilibrative transport).
The facilitated transport is employed by concentration gradient without any energy consumption to drive the process. Active transporters refer to primary or secondary carriers and can deliver molecules against the concentration gradient.
Primary transporters (e.g. ABC transporters) use the energy of the hydrolytic reaction of adenosine triphosphate (ATP) and efflux the substrates from a cell against the concentration gradient, while secondary transporters deliver molecules across the BBB in antiport or symport of ions such as H+, Na+, K+, Cl-, etc. (O'Kane et al., 2004, Dallas et al., 2006). The uptake of molecules via transporters is a temperature-dependent and saturated process following Michaelis-Menten kinetics. In addition, the process can be also affected by competitive (competing for substrate binding), non-competitive (modulating binding to substrate allosterically), uncompetitive (hindering conformational modification of the complex “substrate-transporter”) or mixed-type inhibitors (Krupka, 1983).
The carrier-mediated uptake of different essential nutrients such as amino acids, vitamins, glucose and nucleosides occur across the BBB to keep the brain homeostasis. The same transporters can facilitate the influx of therapeutic compounds. In addition, the efflux transporters can facilitate transport of the drugs from the brain to the plasma, which results in low brain uptake of the drugs, substrates of the efflux transporters. The examples of the transporters expressed at the BBB, their location and substrate specificity are presented in Table 2. The BBB transporters include solute carrier (SLC) family represented by facilitative glucose transporter 1 (GLUT1/SLC2A1), L-type amino acid transporter 1 (LAT1/SLC7A5), monocarboxylic acid transporter 1 (MCT1/SLC16A1), organic anion transporter 3 (OAT3/ SLC22A8), organic anion transporting polypeptide 1A2 (OATP1A2/
SLCO1A2). The efflux transporters are represented by important family of ATP binding cassette (ABC) transporters including P-glycoprotein (P-gp, MDR1/ABCB1), breast cancer-resistance protein (BCRP/ABCG2), and multidrug resistance-associated protein 4 (MRP4/ABCC4), which are considered to be a major obstacle for brain delivery of drugs (Uchida et al., 2011). In addition, it is important to remember that transporters expressed not only at the BBB, but also at the membrane of brain parenchymal cells and BCSFB and can influence elimination and distribution of the drugs in the brain.
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Table 2. The example of transporters, which protein expression has been quantified at the human BBB and their substrates (Ohtsuki and Terasaki, 2007; Uchida et al., 2011)
Gene name
ABCG2 (BCRP) Luminal site glutathione, folic acid, mitoxantrone, topotecan,
dantrolene
8.14 ± 2.26
ABCB1 (P-gp, MDR1) Luminal site vincristine, quinidine,
verapamil 6.06 ± 1.69
ABCC4 (MRP4) Luminal site E217βG, methotrexate,
topotecan 0.195 ± 0.069
sites L-Lactate, monocarboxylates 2.27 ± 0.85
2.3.3 Vesicular trafficking
For normal brain functioning, the transport of large molecular weight compounds such as proteins and peptides is required. These large-molecular cargos can be transported across the BBB via receptor-mediated transport (RMT) or non-specific routes such as cationization and adsorptive-mediated transcytosis (AMT) (Fig. 2).
The RMT involves the formation of the vesicles carrying the molecules across the BBB exemplified by nutrients such as insulin and leptin transported via insulin and leptin receptors, respectively (Duffy and Pardridge, 1987; Golden et al., 1997). The large molecule interacts with a corresponding receptor at the apical side of the BBB cell plasma membrane followed by initiation of the endocytosis process by invagination of receptor-molecule complex and formation of the intracellular trafficing vesicles (Brown and Greene, 1991). The process is finalized by sorting and transporting the vesicles containing the receptor-molecule complex to the basolateral side of the polarized membrane of the endothelial cell, where the release occurs
without disturbance of the barrier properties. In addition, the vesicle can be reversed back to the luminal membrane of the endothelial cells followed by release of molecule into the blood. Alternatively, the vesicle containing the receptor-molecule complex can be transported to lysosomes where it will be degraded. The RMT is a selective process as it requires the initial binding of a molecule to specific receptor present in the plasma membrane of the endothelial cells.
In contrast, the AMT is based on nonspecific electrostatic interactions providing penetration of different cationic proteins such as histone and albumin (Bickel et al., 2001; Herve et al., 2008). The initiation of the process starts when positively charged macromolecular moieties bind to negatively charged plasma membrane of the endothelial cells (Pardridge, 1991). The disadvantage of this transport route is a lack of targeting specificity and possible distribution of compounds to other organs (Bickel et al., 2001).