**3. BBB permeation mechanisms**

Like other cellular membranes in the body, permeation through BBB can occur by passive diffusion, endocytosis and active transport (Diagram 1). Combined effects of the mentioned mechanisms modulate the compound (e.g. Drugs) penetration to the brain.

Diagram 1. Main permeation mechanisms in the brain.

### **3.1 Passive diffusion**

A limited number of drugs and drug like compounds with high lipophilicity and low molecular size can penetrate to the brain mainly by passive diffusion. In order to overcome the surface tension difference between a compound and cellular membrane, physical work is needed and the smaller molecules will need less work. The uncharged forms of the weak acidic and basic compounds have higher permeability rate in comparison with charged molecules in physiologic pH of brain. The charged forms possess hydrophilic characteristics and hydrophilic drugs distribute within blood and cannot cross the endothelial cells and excreted from brain parenchyma. Therefore, the molecules with higher fraction of uncharged form in physiologic pH have higher permeability rate (Fischer et al., 1998). Passive diffusion occurs via two mechanisms (Figure 3):


Like other cellular membranes in the body, permeation through BBB can occur by passive diffusion, endocytosis and active transport (Diagram 1). Combined effects of the mentioned

Passive Diffusion

Free

Bulk phase

Facilitated

Efflux

Influx

Facilitated

A limited number of drugs and drug like compounds with high lipophilicity and low molecular size can penetrate to the brain mainly by passive diffusion. In order to overcome the surface tension difference between a compound and cellular membrane, physical work is needed and the smaller molecules will need less work. The uncharged forms of the weak acidic and basic compounds have higher permeability rate in comparison with charged molecules in physiologic pH of brain. The charged forms possess hydrophilic characteristics and hydrophilic drugs distribute within blood and cannot cross the endothelial cells and excreted from brain parenchyma. Therefore, the molecules with higher fraction of uncharged form in physiologic pH have higher permeability rate (Fischer et al., 1998).

Active transport

Endocytosis



mechanisms modulate the compound (e.g. Drugs) penetration to the brain.

**3. BBB permeation mechanisms** 

Diagram 1. Main permeation mechanisms in the brain.

Passive diffusion occurs via two mechanisms (Figure 3):

**3.1 Passive diffusion** 

BBB permeation

direction.

Fig. 3. Free and facilitated passive diffusion.

#### **3.2 Endocytosis**

In this method, substances (e.g. macromolecules) are engulfed by membrane and pass through the cell by vesicles and release in the other side (Kerns & Di, 2008). Endocytosis occurs via two main methods: bulk phase endocytosis (fluid phase or pinocytosis) and mediated or facilitated endocytosis (receptor and absorptive mediated). Fluid phase endocytosis is a nonsaturable, non-competitive and non-specific method for uptake of extra cellular fluids which is temperature and energy dependent.

Receptor mediated endocytosis facilitates the larger essential molecules uptake selectively using specific receptors present in luminal membrane. Hormones, growth factors, enzymes and plasma proteins are targets for specific receptors (Pardridge, 2007).

Absorptive mediated endocytosis is based on an electrostatic interaction between negatively charged plasma membrane luminal surfaces (glycocalyx which is a negatively charged proteoglycan or glycosaminoglycan) with cationic substances (e.g. peptides) and uptake it in a vesicle into the endothelial cell and release it on the other side (Figure 4) (Ueno, 2009).

This has lower affinity and higher capacity than receptor mediated endocytosis (Alam et al., 2010). Mechanism of vesicle formation (caveolin dependent, dynamin dependent and caveolin- dynamin independent) is not discussed in this chapter and more details could be found in the literature (Lajoie et al., 2010).

Blood Brain Barrier Permeation 9

Essential hydrophilic nutrients (e.g. glucose, amino acids, fatty acids, organic and inorganic ions) reach to brain through influx transporters and receptors. According to the structural similarity of the target drug to the biologic molecules; it can be delivered to the brain using appropriate transporter. Solute carrier family encodes most of the influx transporters which include facilitated, ion coupled and ion exchange transporters that do not need ATP (Eyal et al., 2009). These transporters are responsible for uptake of a broad range of substrates including glucose, amino acids, nucleosides, fatty acids, minerals and vitamins (Alam et al., 2010). The most well studied groups of these bidirectional transporters along with their

Efflux occurs in BBB through both passive and active routes in order to detoxify the brain and prevent from drugs and xenobiotics exposures. There are several kinds of efflux transporters such as ATP binding cassette transporters (ABC), organic anion transport systems, amino acid transport systems and so on (Ueno, 2009). ABC transporters are primary active systems which are responsible for different efflux activities including P-glycoprotein (P-gp), multi-drug resistance proteins (MRPs), and breast cancer related protein (BCRP). P-gp (the most studied ABC transporter), located in luminal side of BBB, immediately pump most of the drugs and xenobiotics back to the blood and decrease the net penetration to the brain. A broad range of drugs, generally including un-conjugated and cationic substances (Table 1) are substrates for P-gp, where some of them are able to inhibit P-gp and lead to increased permeability of coadministered drugs. This fact can be used as a drug delivery strategy to the brain. Along with P-gp, MRPs and BCRP are responsible for main part of drug efflux in BBB and their effect are dependent to their localization and expression level in normal and pathologic conditions. Over expression of these transporters considered as one of the major reasons of pharmacoresistance of brain diseases and their inhibition, bypassing and regulating methods are important for

Existing enzymes in BBB can be regarded as second barrier after negative surface charge. These enzymes involve in disposition of drugs and xenobiotics before entering the endothelial cells of capillaries. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, adenosine tri-phosphatase and nucleoside di-phosphatase are among well studied enzymes

The rate and the extent of drug transport to the brain are needed for drug discovery studies (both peripheral and CNS drugs) and different methods developed in order to study the pharmacokinetic profile of drug candidates. BBB permeability depends on physicochemical properties of drug compound and physiologic functions of the BBB (physical barrier, transport, metabolic pathways) and need special study techniques. These techniques include *in vivo*, *in vitro*, and *in silico* methods (Diagram 2) which are complement in most cases and researchers are able to define different aspects of drug passage to the brain using these

**3.3.1 Influx transporters** 

**3.3.2 Efflux transporters** 

properties and activities are summarized in Table 1.

CNS drug development (Loscher & Potschka, 2005).

**4. BBB permeation measurement methods** 

**3.4 Metabolism in BBB (Enzymatic barrier)** 

distributed within BBB (Ueno, 2009).

methods.

Fig. 4. Bulk phase and facilitated endocytosis.

#### **3.3 Active transport**

Hydrophilic drugs which cannot penetrate the brain through passive diffusion and lipophilic drugs which cannot penetrate the brain, in contrast of their suitable characteristics for BBB permeation are substrate for drug transporters of the BBB. Also some compounds are substrates for transporters and at the same time they are delivered by passive diffusion or endocytosis. Drug transporters are integral membrane proteins which is able to carry the drug usually against the concentration gradient into and out of the cell.

The overall exposure of xenobiotics to brain through these transporters depends on their location and expression level according to the normal and pathophysiologic conditions. Two types of drug transporters according to their driving forces (ATP dependent and ATP independent) are known. Active transporters broadly categorized as primary (ATP dependent), secondary or tertiary (ATP independent) (Murk et al., 2010).

There are two types of transporters:


Hydrophilic drugs which cannot penetrate the brain through passive diffusion and lipophilic drugs which cannot penetrate the brain, in contrast of their suitable characteristics for BBB permeation are substrate for drug transporters of the BBB. Also some compounds are substrates for transporters and at the same time they are delivered by passive diffusion or endocytosis. Drug transporters are integral membrane proteins which is able to carry the

The overall exposure of xenobiotics to brain through these transporters depends on their location and expression level according to the normal and pathophysiologic conditions. Two types of drug transporters according to their driving forces (ATP dependent and ATP independent) are known. Active transporters broadly categorized as primary (ATP

1. Carrier mediated transporters which express on both the luminal and abluminal membranes and operates in both blood to brain and brain to blood directions. 2. Active efflux transporters which mediate extruding drugs and other compounds from brain (Alam et al., 2010). Although the main role of the drug transporters is carrying the drugs and other xenobiotics into and out of the brain but they are responsible for other cell processes such as inflammation, differentiation of immune cells, cell detoxification, lipid trafficking, hormone secretion and development of stem cells

drug usually against the concentration gradient into and out of the cell.

dependent), secondary or tertiary (ATP independent) (Murk et al., 2010).

Fig. 4. Bulk phase and facilitated endocytosis.

There are two types of transporters:

(Murk et al., 2010).

**3.3 Active transport** 

#### **3.3.1 Influx transporters**

Essential hydrophilic nutrients (e.g. glucose, amino acids, fatty acids, organic and inorganic ions) reach to brain through influx transporters and receptors. According to the structural similarity of the target drug to the biologic molecules; it can be delivered to the brain using appropriate transporter. Solute carrier family encodes most of the influx transporters which include facilitated, ion coupled and ion exchange transporters that do not need ATP (Eyal et al., 2009). These transporters are responsible for uptake of a broad range of substrates including glucose, amino acids, nucleosides, fatty acids, minerals and vitamins (Alam et al., 2010). The most well studied groups of these bidirectional transporters along with their properties and activities are summarized in Table 1.

#### **3.3.2 Efflux transporters**

Efflux occurs in BBB through both passive and active routes in order to detoxify the brain and prevent from drugs and xenobiotics exposures. There are several kinds of efflux transporters such as ATP binding cassette transporters (ABC), organic anion transport systems, amino acid transport systems and so on (Ueno, 2009). ABC transporters are primary active systems which are responsible for different efflux activities including P-glycoprotein (P-gp), multi-drug resistance proteins (MRPs), and breast cancer related protein (BCRP). P-gp (the most studied ABC transporter), located in luminal side of BBB, immediately pump most of the drugs and xenobiotics back to the blood and decrease the net penetration to the brain. A broad range of drugs, generally including un-conjugated and cationic substances (Table 1) are substrates for P-gp, where some of them are able to inhibit P-gp and lead to increased permeability of coadministered drugs. This fact can be used as a drug delivery strategy to the brain. Along with P-gp, MRPs and BCRP are responsible for main part of drug efflux in BBB and their effect are dependent to their localization and expression level in normal and pathologic conditions. Over expression of these transporters considered as one of the major reasons of pharmacoresistance of brain diseases and their inhibition, bypassing and regulating methods are important for CNS drug development (Loscher & Potschka, 2005).

#### **3.4 Metabolism in BBB (Enzymatic barrier)**

Existing enzymes in BBB can be regarded as second barrier after negative surface charge. These enzymes involve in disposition of drugs and xenobiotics before entering the endothelial cells of capillaries. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, adenosine tri-phosphatase and nucleoside di-phosphatase are among well studied enzymes distributed within BBB (Ueno, 2009).

#### **4. BBB permeation measurement methods**

The rate and the extent of drug transport to the brain are needed for drug discovery studies (both peripheral and CNS drugs) and different methods developed in order to study the pharmacokinetic profile of drug candidates. BBB permeability depends on physicochemical properties of drug compound and physiologic functions of the BBB (physical barrier, transport, metabolic pathways) and need special study techniques. These techniques include *in vivo*, *in vitro*, and *in silico* methods (Diagram 2) which are complement in most cases and researchers are able to define different aspects of drug passage to the brain using these methods.

Blood Brain Barrier Permeation 11

Intra venous ART, PET, … In situ perfusion

Intra cerebral micro dialysis

Brain derived cell cultures

Co cultures

Primary brain epithelial cell cultures

Epithelial cell cultures (Caco2)

Modified

epithelial PAMPA cell cultures

Non brain derived cell lines

Linear

Non linear

Linear

Non linear

IAMs

Intra carotid

Cell based

Ex vivo

Non cell based

Statistical

Mechanistic

The drug is available in blood in the free (unbound) and bounded (protein bounded, erythrocyte bounded, tissue bounded) forms. The unbound drug molecules equilibrate across the BBB and brain. The spaces that these equilibria occur are: blood, interstitial fluid, intercellular and intracellular fluids. Figure 5 shows these equilibria schematically. The speed of the equilibria to reach the steady state define the rate of drug distribution within brain, and the slowest one would be the rate limiting step. For poor CNS penetrantes, the BBB permeation or the diffusion of drug molecules within the brain tissue is the rate limiting step. Total brain concentration which allow us just to rank drug candidates according to their CNS total levels and general CNS penetrability can be measured using most of the *in vivo* methods, while there is just a few methods which are

Diagram 2. Brain drug testing methods.

able to provide free fractions directly.

**4.1.1 Bound and unbound drug concepts** 

 In vivo

 In vitro

 In silico

**4.1 BBB permeation data** 

BBB measurement methods


Table 1. Some of the well studied influx and efflux transporters of brain.

Anionic drugs and nucleotides Benzylpenicillin,

**nutrients** 

Methotrexate

Mercaptopurine, Methotrexate, Valproic

Cimetidine ,

Pregabalin

Simvastatin,

γ- Hydroxybutyrate

Cl-, Na+, K+, H+, HCO3-

Anti cancer and anti HIV

Some anti cancer Drugs Efflux

Anti cancer drugs,

corticoids

Drugs

Adenosine Influx

acid

Fexofenadine, Digoxin,

Valacyclovir, Zidovudine,

Desipramine, Metformin, Amantadine, Memantine, Cisplatine, Quinin

L-phenylalanine, Ltyrosine, L-tryptophan, L-lucine, Levodopa, -Methyldopa, Baclofen, Melphalan, Gabapentin,

**Influx/ Efflux** 

Influx

Influx

Influx / Efflux

Influx / Efflux

Influx

 Influx / Efflux

Efflux

Efflux

**Transporter name Substrates Sample drugs and** 

Anionic amphipathic molecules with molecular weight greater than 450 Daltons and a high degree of albumin binding

Bidirectional transport of small hydrophilic positively charged

neutral amino acids with branched or aromatic side

Purine and pyrimidine

xenobiotics (normally unconjugated, cationic substances)

Drugs and xenobiotics (normally conjugated, anionic

Drugs and xenobiotics (overlap with P-glycoproteins and multidrug resistance proteins)

Table 1. Some of the well studied influx and efflux transporters of brain.

HMG-CoA reductase inhibitors that contain a carboxylic acid

Hexose transporters Hexose nucleosides Glucose Influx

compounds

System L. Bidirectional transport of large

chains

moiety

nucleosides

Ion transporters Bidirectional transport of small ions

substances)

P-glycoproteins A broad range of drugs and

Organic anion transporting polypeptides

Organic anion transporters

Organic cation transporters

Monocarboxylate transporters

Nucleoside transporters

Multi-drug resistance proteins

Breast cancer resistant proteins

Diagram 2. Brain drug testing methods.
