Drug Transporters at the Blood–Brain Barrier

CHAPTER 5 Drug Transporters at the Blood–Brain Barrier DAVID DICKENS,* STEFFEN RADISCH AND MUNIR PIRMOHAMED Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK *Email: [email protected] 5.1 The Blood–Brain Barrier 5.1.1 Overview The central nervous system (CNS) is of fundamental importance for the control and regulation of physiological processes. It is an extremely sensitive microenvironment and strict homeostatic regulation is essential in order to maintain neuronal signalling. A key aspect in regulation is the physical separation of the CNS from the rest of the body by means of CNS barriers, which restrict the access and exit of molecules to and from the brain. This provides control over the concentration and composition of ions, neurotransmitters, macromolecules, neurotoxins and nutrients, as well as the entry/exit of xenobiotics.1 Three cellular barriers enclose the CNS:  The blood–brain barrier (BBB), which separates the blood and the brain interstitial fluid;  The choroid plexus (CP), which is between the blood and ventricular cerebrospinal fluid (CSF); RSC Drug Discovery Series No. 54 Drug Transporters: Volume 1: Role and Importance in ADME and Drug Development Edited by Glynis Nicholls and Kuresh Youdim r The Royal Society of Chemistry 2016 Published by the Royal Society of Chemistry, www.rsc.org 151 152 Chapter 5  The arachnoid epithelium (meningeal barrier), which separates the blood and the subarachnoid CSF. These three cellular barriers regulate the exchange of compounds and nutrients at the interfaces between the blood and the brain or its fluid spaces.2 The BBB can be considered the most important of the three and has been studied extensively for its role in pharmacology and development, mainly because it has the biggest surface area for the passage of compounds from the blood into the brain, and it directly separates the blood from the extracellular fluid (ECF) of CNS neuronal tissue, and hence is in close proximity to neurons. Due to this, the primary focus of this chapter will be on the BBB with the CP briefly discussed. The BBB is a cellular barrier that comprises specialised brain endothelial cells that form the walls of microcapillaries and can regulate the passage of endogenous substances and xenobiotics into and out of the brain, which maintains brain homeostasis and neuronal signalling (Figure 5.1). The barrier is not static and can be considered a biologically active interface with regulatory actions that involve transport, secretory and enzymatic roles.3 This can thus present both a challenge and an opportunity for drug development. The present chapter outlines the role of drug transporters at the BBB and CP in drug pharmacology, with a particular focus on the distribution and penetration of drugs into the brain. 5.1.2 BBB in Numbers The human brain is composed of approximately 86 billion neurons with similar numbers of glial cells that utilise between 15 and 20% of the entire energy of the body.4 To provide this energy, the human BBB has a large surface area of between 15 and 25 m2 for the passage of nutrients. In addition, the cell body of a neuron is between 10 and 20 mm from the nearest microcapillary, providing a network that infuses the entire brain.1 This dense network results in a microvessel length of 600 km, with the diameters of microcapillaries being as small as 7–10 mm. The BBB vasculature is 3% of the total brain volume with the brain endothelial cells comprising 0.1% of the total brain by cell number.5 The scale of the BBB results in 15–20% of the blood flow from the heart going to the brain. The size and importance of the BBB in maintaining homeostasis of the brain requires regulation with associated cells, so a cellular framework is formed that has been coined the ‘neurovascular unit’.6 5.1.3 Neurovascular Unit The BBB is not only composed of brain endothelial cells; other cell types also make up the neurovascular unit. The brain endothelial cells form the physical barrier of the microvessel with contributions from pericytes, astrocytes and neurons2 (Figure 5.1). Drug Transporters at the Blood–Brain Barrier Figure 5.1 153 The BBB as a neurovascular unit. The BBB forms a cellular framework termed the neurovascular unit.6 The brain endothelial cells comprise the cell type that forms the physical barrier of the microvessels within the BBB. Pericytes at the BBB are involved in regulating cerebral blood flow while astrocytes regulate BBB functions, including transporter localisation and tight junction proteins. The brain endothelial cells are a highly specialised endothelial cell type due to their interactions with surrounding CNS cells and tissues during development and maturation. During development, early specialisation of cells into endothelial cells is due at least in part to the excretion of retinoic acid from glial cells.7 Additionally, neuronal progenitor cells, via the WNT/ b-catenin signalling pathway, have been proposed to drive the endothelial progenitor cells towards the specialised brain endothelial cell phenotype.8 Surrounding cell types such as pericytes and astrocytes assist in maintaining this specialised phenotype into adulthood. Pericytes are a type of perivascular cell that wraps around endothelial cells, with the highest density found in vessels of the neural tissues. As well as 154 Chapter 5 sheathing brain endothelial cells, pericytes of the CNS are attached to the same basement membrane.9 They have a number of important roles at the BBB, including developmental, regulation of cerebral blood flow and maintenance of BBB permeability.10–13 Brain pericytes contribute to the developing BBB by controlling tight junction formation and by decreasing the expression of factors that are involved in increasing permeability.10 In the mature BBB, the physical coverage of brain endothelial cells by pericytes and the regulation of expression of specific factors, such as the transmembrane protein MFSD2, control BBB permeability and integrity.10,13 The cerebral blood flow can be altered following neuronal activity via the release of glutamate, which results in the loosening of pericytes and thus dilation of brain capillaries.12 Astrocytes are the most abundant type of cell in the human brain and can be involved in regulating BBB functions such as tight junction proteins and transporter localisation.2 For example, astrocytes secrete a sonic hedgehog protein whose corresponding receptors expressed on brain endothelial cells promote BBB formation and integrity during embryonic development and adulthood.14 The astrocytes have end feet that provide almost complete coverage (499%) of the abluminal cell membranes of the brain endothelial cells, thus providing a physical interaction between astrocytes and the BBB.15 5.1.4 Physical Barrier The brain endothelial cells of the BBB form a physically tight barrier by the expression of tight junction and transporter proteins. This means the endothelial cells at the BBB have a specialised phenotype compared with peripheral endothelial cells. This is exemplified by measures of electrical resistance that are used as an indirect measure of barrier tightness. The electrical resistance of the BBB in vivo is thought to be at least 1500 O cm2, with values up to 6000 O cm2 recorded, and a recent in vitro model of human brain endothelial cells derived from induced pluripotent stem cells (iPSCs) has achieved values as high as 6000 O cm2.16,17 This is much higher than the resistance observed in peripheral endothelial cells; for example, in small bowel and muscle endothelial capillaries, the resistance values are 2 O cm2 and 20 O cm2, respectively.18,19 This tightness of the BBB results in a low paracellular permeability. The tight junctions between the brain endothelial cells are considered essential for both conferring the tightness of the BBB, thus reducing paracellular permeability, and in polarising the brain endothelial cells into a luminal and abluminal membrane.2 The tight junctions are composed of a complex of proteins (including zonula occludens (ZO)-1, occludin, claudin 3, claudin 5 and claudin 121) that span the intercellular cleft and thereby constitute a physical barrier. They are part of the junctional complex generally involved in cell adhesion. The junctional complex also includes adherens junctions, which are comprised of important structural proteins such as the cadherin–catenin complex. These attach endothelial or epithelial 155 Drug Transporters at the Blood–Brain Barrier 1,20 cells and are essential for the formation of tight junctions. Adherens junctions are located underneath tight junctions at the most apical part of the junctional complex between the apical and basolateral cell membrane.21 Tight junctions also contain junctional adhesion molecules (JAMs), which are proposed to be involved in leukocyte cell adhesion.1,22 In addition, scaffold proteins such as ZO-1, ZO-2 and ZO-3 cluster the spanning proteins and connect them to the actin/myosin cytoskeleton.23 Tight junctions can also restrict membrane trafficking within one cell, thereby separating the apical from the basolateral membrane domain.21 Figure 5.2 illustrates the basic composition and arrangement of tight junctions and adherens junctions. Figure 5.2 Model of BBB tight junctions and adherens junctions. A simplified model illustrating tight junction and adherens junction composition and arrangement. JAMs, claudin 3/5 and occludin are important tight junction proteins with the claudins and occludins spanning and sealing the intercellular cleft between brain endothelial cells. ZOs comprise scaffolding protein clusters and connect tight junction proteins to the actin/myosin cytoskeleton. Vascular endothelial (VE)-cadherin proteins are adhesive junction proteins important for structural integrity and tight junction formation. They are linked to the cytoskeleton by means of the catenin scaffolding proteins. 156 5.1.5 Chapter 5 Transport at the BBB Compounds can enter the brain by a number of different mechanisms: the paracellular aqueous pathway, transcellular lipophilic pathway, transportermediated, receptor-mediated transcytosis and adsorptive transcytosis (Figure 5.3). The focus of this chapter is on the role of transporters in Figure 5.3 Model of BBB transport. Simplified model illustrating important drug transport mechanisms across brain endothelial cell layers. (A) Passive diffusion: in the case of transcellular diffusion, the accepted rule is that the higher the lipophilicity of a compound, the greater the passive diffusion through the BBB. (B) Adsorptive and receptor mediated transcytosis: proteins such as insulin and albumin can be transported through the BBB by specific receptor-mediated endocytosis and transcytosis pathways. (C) ABC transporter efflux: carrier-mediated efflux is a major obstacle for many pharmacological agents as it is a mechanism involved in expelling drugs from the brain. (D) SLC transport: secondary-active and facilitative transporters of the SLC transporter family can be involved in the carriermediated uptake and removal of compounds into and out of the brain. Drug Transporters at the Blood–Brain Barrier 157 affecting the transcellular pathway by efflux or influx mechanisms, and how this affects drug delivery and/or prevents exposure to the brain. The importance of the transcellular lipophilic pathway in the passage of drugs through cells has been hotly debated in a number of recent reviews and will not be discussed further in this chapter. Interested readers are referred to various in-depth reviews concerning this debate on the relative significance of transporter-mediated and passive diffusion of compounds into cells.24,25 However, it should be noted that lipophilicity is not always a good guide to CNS penetration, as even highly lipophilic drugs can be prevented from entry by efflux transporter activity.26 Increased lipophilicity can also lead to disadvantages such as increased non-specific binding.27,28 The brain endothelial cells are polarised and express a range of efflux and influx transporters at the apical and basolateral membranes that control the movement of substances through the cells. They are known to have more mitochondria than peripheral endothelial cells (such as skeletal endothelial cells), probably due, at least in part, to the active nature of the barrier and an increased energy requirement for active transport processes.29,30 Therefore, through the action of transporters, and by providing a physical barrier, the BBB aids in keeping out toxins from the brain while allowing in essential nutrients to maintain brain homeostasis. From a pharmacological perspective, it has been reported that it prevents 498% of small compound drugs and nearly all large-molecule therapeutics (biologicals) from having a pharmacological effect on the brain.28 5.2 Modelling of the BBB Modelling of the BBB by in vitro and in vivo methods is an important endeavour for predicting transport of drugs at the human BBB, as each cellular barrier has its own novel characteristics and transportisome. There have been recent advances in the study of drug transport in BBB models, and this section will outline the use and relevance of these different in vivo, in vitro and in silico methods. All of the models outlined below have both advantages and limitations, with systems biology/physiologically-based modelling approaches that interpolate transport data from multiple laboratory-based models being particularly useful. For example, the prediction of in vivo penetration of CNS drugs can be improved by integrating permeability, P-glycoprotein (P-gp) efflux and drug free fractions in the blood and brain into a mathematical model.27,31 Alternatively, new molecular entities can be modelled at an early stage using quantitative structure–activity relationships (QSAR) for both biological activity and BBB permeability, taking into account interactions with both the target receptor and BBB transporters32,33 (covered in more detail in Chapter 7). 5.2.1 Cellular Models of the BBB Cellular in vitro models of the BBB have improved in recent years and can provide a useful tool for investigating drug transport. A number of brain 158 Chapter 5 immortalized endothelial cell lines have been developed and include human (hCMEC/D3), rat (RBE4 cells) and mouse (endo3) cell lines. The hCMEC/D3 cell line is a well-characterised human brain endothelial cell line34,35 that has been used for in vitro drug uptake assays by multiple groups to study efflux and influx transporters.36–40 However, like all cell lines generated to date, these brain endothelial cells have lost some of their unique protein expression pattern outside of their native environment and thus display a more generic endothelial cell phenotype.41 This leads to a loss of barrier tightness and consequently transcellular permeability studies of the compound of interest are not possible, which remains a major drawback of this cell line. However, cell lines that do provide the possibility to study transcellular permeability include cells such as Caco-2 and MDCK transfected with P-gp, although they are of epithelial origin and so their relevance to the BBB is highly debatable.42 Primary brain endothelial cells provide a useful compromise between the cell line approach and in vivo experiments.43 However, they are low to medium throughput due to the fact that only 0.1% of brain cells are endothelial cells, and thus yields are low and extended passaging results in a loss of phenotype. Porcine, bovine and rodent models are favoured sources of primary brain endothelial cells and, in co-culture with astrocytes, high transendothelial electrical resistance (TEER) is achievable—up to 1300 O cm2. This high electrical resistance means a tight barrier is formed and thus the models can be useful as a tool for drug permeability studies investigating transcellular permeability through the BBB.44 Recent developments include deriving brain endothelial cells from stem cell sources.8,17,45,46 The most developed model utilises either iPSCs or embryonic stem cells (ES) to derive cells with a brain endothelial phenotype with a high TEER (around 5000 O cm2), and as such the cells are suitable for transcellular permeability studies.8,17 This is an exciting area for further research as it could in the longer term lead to an in vitro BBB model derived from patient cohorts of responders and non-responders to a particular drug treatment, to investigate whether transport is a variable for drug response. 5.2.2 In vivo Models A variety of in vivo models exist to study the transport of drugs at the BBB. These range from non-mammalian organisms such as zebrafish through to rodents and humans. The non-mammalian studies are at an early stage, but proof-of-principle drug transport studies have been performed. In fruit flies (Drosophila melanogaster), a P-gp homologue (Mdr65) has conserved function at its CNS barrier that confers protection against cytotoxic pharmaceuticals,47 and evidence from zebrafish suggests that drug transport could also be investigated in this model.48,49 Rodents are the most common in vivo animal model used to investigate drug transport at the BBB, in whole animal studies or with techniques such as microdialysis or in situ perfusion. In situ perfusion entails controlling the Drug Transporters at the Blood–Brain Barrier 159 circulation to the brain by directly infusing saline and drug into the major vessels leading to the brain. The amount of drug in the brain is determined at set time points and, from these measurements, the kinetics and permeability constants of brain uptake are determined.50 Microdialysis determines the free drug concentration in the brain by the insertion of a probe with a semi-permeable membrane into the rodent brain and is one of the most sensitive methods available for studying BBB transport of drugs in vivo.43 The amount of free drug is the concentration of drug that is unbound while the total is the combined protein-bound and unbound drug concentration. However, the insertion of the probe can lead to damage of the surrounding tissue, including the BBB.51 In whole animal experiments using rodents, the total brain concentration of a drug can be determined following administration. The use of knockout mice to determine the effect of a specific transporter in the uptake of a compound into the brain has become an important technique for the study of BBB transport. Due to the recent advances in generating and culturing rat ES lines with homologous recombination,52 knockout rats such as Mdra1a / and Abcg2 / are now being used for drug transport studies, for example in determining the brain to plasma ratios of novel small compound drugs.53 A hypothesised disadvantage of knockout animals is that compensatory processes could induce other drug transporters to be upregulated following knockout of a particular transporter. However, in a study by Agarwal et al. in Mdr1a/b / or Abcg2 / rodent knockout animals, no difference was observed in the expression of the measured proteins (29 protein molecules, including 12 ABCs, 10 SLCs, 5 receptors and 2 housekeeping proteins) in the brain capillary endothelial cells of the single and double knockout animals.54 Nonetheless, it should also be noted that species differences in transporter expression need to be taken into account when extrapolating data to humans.55 Positron emission tomography (PET) is a non-invasive technique that enables the regional brain measurement of radioactivity for a radiolabelled compound to be obtained in both animals and humans56 (refer to Drug Transporters: Volume 2 Recent Advances and Emerging Technologies, Chapter 6 for further details). For example, a study in mice showed that elacridar increased the brain uptake of radiolabelled gefitinib (a tyrosine kinase inhibitor) by around 12-fold compared with control mice receiving gefitinib only.57 Another avenue is the use of a fluorescent probe substrate and bioluminescence imaging, for example D-luciferin, the endogenous substrate of firefly luciferase, has recently been shown to be a breast cancer resistance protein (BCRP) substrate.58 This approach requires a transgenic mouse but is the first study to generate and validate a specific BCRP probe for imaging studies. Sampling from the CSF in humans can be used as a surrogate for the brain ECF concentration59 and this approach has been used to obtain the unbound drug concentrations of some P-gp substrates in rodents.60 However, due to the differences in transporter expression and activity at the BBB compared with the CP, the CSF does not always correlate with the brain ECF 160 Chapter 5 61,62 drug concentrations. One reason for this may be that drug transport into the CSF takes place mainly at the CP, with only a small part from the flow of the brain fluid emptying into the CSF1 (see Section 5.5). 5.3 Efflux Transporters Expressed at the BBB The family of ATP binding cassette (ABC) transporter genes currently comprises 48 genes (excluding pseudogenes). The official list is available on the HGNC website (http://www.genenames.org/cgi-bin/genefamilies/set/ 417). The ABC superfamily is further divided into seven subfamilies with designated letters A–G and each gene assigned a unique number following the family root and the subfamily letter, e.g. ABCB1. All ABC transporters, corresponding to their name, are characterised by the presence of two ATP binding cassettes, also referred to as the nucleotide binding domains (NBDs). NBDs bind to ATP and the energy derived from ATP hydrolysis is utilised as the driving force for active substrate translocation against an electrochemical gradient. NBDs typically consist of two highly conserved nucleotide binding motifs, referred to as Walker A and Walker B, linked by another highly conserved motif, the ABC signature or C motif.63 In addition to the NBDs, ABC transporters further comprise two transmembrane domains (TMDs) with varying numbers of transmembrane helices (TMHs). While a functional transporter consists of the core structure of two TMDs and two NBDs, the corresponding gene may only encode a half-transporter, with one TMD and one NBD, and subsequent homo- or hetero-dimerisation at the protein level.63 Functionally, ABC transporters are involved in the unidirectional, active extrusion of xenobiotics and endogenous substances, such as metabolic products and lipids, and are thus important in cell detoxification systems.64 A number of recent reviews have suggested that too much focus has been placed on BBB permeability and not enough on understanding BBB transport, however this view is debated.59,65 Along with this clearance function, many ABC transporters are recognised as mediators of a multidrug resistance phenotype, particularly for cytotoxic anticancer drugs such as vinblastine and doxorubicin.66 The ABC efflux transporters known to be expressed at the BBB are outlined in Figure 5.4 and discussed further below. 5.3.1 P-gp P-gp (ABCB1, MDR1) is the prototypical efflux transporter. It is an ATPdependent multidrug efflux transporter that is highly expressed at the BBB and can affect the permeation of its substrate drugs into the brain.67 P-gp is localised to the luminal side of the brain endothelial cell and as such pumps drugs out of the cells and into the blood. This reduces penetration into the CNS and has led to the recognition of its role as a ‘‘gatekeeper’’. This is because P-gp can act as a protective mechanism against potentially toxic xenobiotics.26 This gatekeeper role is enhanced by its broad substrate base, Drug Transporters at the Blood–Brain Barrier Figure 5.4 161 Subcellular localisation of efflux transporters at the BBB. Human ABC transporters at the BBB that have been introduced in this chapter with confirmed subcellular localisation are P-gp (ABCB1), BCRP (ABCG2) and MRP4 (ABCC4) (in red). In blue is the proposed localisation of efflux transporters that could be putatively expressed at the BBB, MRP1 (ABCC1) and MRP5 (ABCC5). and readers are referred to Schinkel and Fromm for extensive reviews on this topic.26,68 The topology and structure of P-gp have been well described, which has led to a functional understanding of its poly-specific drug binding. Utilising X-ray crystallography techniques, the structure of P-gp from a variety of non-human organisms has been solved.69–72 As ABCB transporters have the same basic core structure of two TMDs and two NBDs, the mechanistic insights gained from these crystal structures are of high value for the whole ABCB transporter family. Mouse MDR1A was crystallised in the inward-facing conformation and the two TMDs displayed a pseudo two-fold symmetry to each other, forming a large portal with many hydrophobic amino acid residues localised in the membrane at the assumed substrate binding pocket.69 Consistent with the predicted P-gp topology, each domain displays a bundle of six TMHs.69 Mechanistically, the inward-facing structure does not allow substrates to access the binding pocket from the outer membrane layer or the extracellular space. Instead, it has been suggested that substrates enter the binding pocket from within the inner membrane layer, which further stimulates ATP binding to the NBDs and is followed by a conformational change of P-gp to the outward-facing state.69 ATP hydrolysis has been suggested to be a likely mechanism to disrupt the dimerisation of both NBDs, and to allow the transporter to flip back into the inward-facing conformation.69,73 This provides an example of how translation of structure 162 Chapter 5 to function using structural biology techniques can address mechanistic questions of how a transporter functions at the molecular level. Due to the broad substrate specificity of P-gp, a number of drugs from several different chemical classes are substrates of this transporter.68 A variety of methods have been utilised to investigate the importance of P-gp at the BBB, including overexpression using in vitro cell models, knockout animals such as Mdr1a / , isolated brain endothelial cells and chemical inhibitors of P-gp,68 as described in Sections 5.2.1 and 5.2.2. In rodents, P-gp is encoded by the Mdr1a and Mdr1b genes. The work by Schinkel et al. in 1994 was fundamental in showing the importance of P-gp at the BBB,26,67 indicating how both the pharmacokinetics and toxicity of a drug were affected by knockout of mouse Mdr1a. Many examples are available to demonstrate the effect of P-gp on drug penetration. During HIV infection, the brain is considered to be a sanctuary site for the virus. Therefore, adequate concentrations of anti-retroviral drugs would have to be achieved in the brain in order to attempt to eliminate the virus. However, many of the protease inhibitors used in HIV treatment, such as saquinavir, indinavir and nelfinavir, are P-gp substrates, which restricts their access to the brain. This contributes to viral persistence and reduced effectiveness. The importance of P-gp for these compounds was shown using the Mdr1a / knockout mouse in which, following intravenous injection, the brain concentration of drug was elevated from 7- to 36-fold relative to control mice.74 To overcome the action of P-gp, a number of approaches have been tried and include the use of inhibitors of P-gp and pro-drug approaches.75–77 Imatinib is a tyrosine kinase inhibitor used in the treatment of multiple cancers, including chronic myelogenous leukaemia (CML). Using a number of approaches, including the use of multidrug resistant cell lines, transcellular permeability in P-gp overexpressing cells and Mdr1a/1b / knockout mice, imatinib has been shown to be a P-gp substrate.78–81 In one study, an 11-fold greater uptake of imatinib into the brain was observed in the Mdr1a/1b / knockout mouse compared with control mice, providing an explanation for one of the limitations of using imatinib in CML patients, who can show a complete haematological response to the drug but still retain a sanctuary site in the CNS.78–81 In contrast, the restriction of BBB drug entry by P-gp can sometimes be of benefit, since it may result in a reduced CNS side effect profile and for some drugs can result in a repositioning of the drug to another clinically therapeutic area. An example of this is domperidone, a dopamine antagonist with limited CNS effects (due to its P-gp mediated transport at the BBB67) that can be used in patients with Parkinson’s disease in order to minimise adverse effects on the extrapyramidal system, i.e. involuntary movements. Similarly, the P-gp substrate loperamide, an opioid, is excluded from the CNS, and as such its peripheral opioid-like effects can be used clinically as an antidiarrhoeal.26 Colchicine is a potentially neurotoxic alkaloid used as an anti-gout medication that is excluded from the brain on oral dosing due, at least in Drug Transporters at the Blood–Brain Barrier 163 part, to its efflux at the BBB by P-gp. Studies measuring colchicine uptake into the brain of rodents in the absence or presence of a P-gp inhibitor (PSC-833), using microdialysis and in situ perfusion methodologies, indicate enhanced brain uptake in the presence of PSC-833.82–84 Notably, when directly injected into the brain of rodents to bypass the effect of P-gp, colchicine induces neurotoxicity, leading to sporadic dementia in the animals.84 5.3.2 BCRP BCRP (ABCG2) is a drug efflux transporter that is part of the ABCG subfamily. BCRP was discovered and cloned from a multidrug resistance breast cancer cell line and found to extrude a number of chemotherapeutic drugs.85 Together with P-gp, BCRP is recognised as a mediator for multidrug resistance in cancer, with a complementary and overlapping substrate profile to P-gp.85 Unlike P-gp, the BCRP gene encodes only one TMD and one NBD with oligomerisation of the protein being critical to produce a functional transporter.86 As well as being expressed in breast cancer, BCRP has been found to have a functional role in a number of tissues, including the BBB.87 It displays apical membrane localisation, with immunofluorescence studies and proteomic analysis confirming its brain endothelial cell expression at the luminal membrane.88–90 BCRP substrates include both endogenous and xenobiotic compounds. Endogenous BCRP substrates include urate and 17b-estradiol-17-b-Dglucuronide (E217bG), with xenobiotic compounds including the chemotherapeutic drug mitoxantrone, the H2 blocker cimetidine and the HMG-CoA reductase inhibitor pitavastatin.87 One of the first studies showing the importance of BCRP at the BBB investigated the transport of mitoxantrone in Mdr1a / knockout mice in the presence and absence of a dual BCRP and P-gp inhibitor (elacridar).91 Real-time quantitative reverse transcription polymerase chain reaction (PCR) was used to show a high expression of BCRP in brain endothelial cells compared with the cortex in wild type mice. However, a number of negative studies at the BBB for known BCRP substrates exist,92 a finding that may be explained at least in part by the overlapping substrate specificity of BCRP and P-gp. BCRP is thought to act in conjunction with P-gp in preventing the entry of some xenobiotics into the brain93 and, for specific co-substrates, both P-gp and BCRP may need to be inhibited or knocked out for a functional inhibitory effect to be observed. The interplay between efflux transporters at the BBB is discussed in more detail in Section 5.3.5. For BCRP, a non-synonymous genetic polymorphism (Q141K) at the NBD has in multiple studies been linked to high serum urate levels and thus to gout94–96 due to reduced urate transport in cells.94 The variant BCRP protein has been linked to reduced stability at the NBD and thus reduced protein expression.95 The relevance of this for BCRP-mediated transport at the BBB is unknown but it should be noted that in patients with diffuse large B-cell 164 Chapter 5 lymphoma, the Q141K polymorphism was associated with chemotherapyinduced diarrhoea following R-CHOP treatment.97 R-CHOP comprises rituximab plus cyclophosphamide/doxorubicin/vincristine/prednisone, with cyclophosphamide and doxorubicin, a BCRP substrate drug, highly associated with diarrhoea.97 5.3.3 MRP4 MRP4 (ABCC4) is part of the multidrug resistance associated protein (MRP; ABCC) subfamily and is highly expressed at the BBB.98 Twelve genes are assigned to the ABCC subfamily of membrane transporters designated as ABCC1–ABCC12 with the encoded proteins being further divided into three classes, namely the MRPs, the sulfonylurea receptors (SURs) and the cystic fibrosis transmembrane conductance regulator (CFTR). The MRP transporters are ATP-dependent transporters with two TMDs and NBDs, and the transport of substrates may be either glutathione (GSH)-independent or GSH-dependent (GSH may either be co-transported with the substrate or it may act as a stimulant99). MRP4 was first identified as a protein that conferred resistance to nucleoside-based antiviral drugs in a T-lymphoid cell line.100 It is known to display overlapping but also quite distinct transport characteristics within the ABCC subfamily. A number of substrates of both endogenous and xenobiotic origin have now been identified for MRP4. Prostaglandins are a unique endogenous substrate of MRP4.101 They are effluxed by MRP4 and an in vitro study determined that nonsteroidal antiinflammatory drugs (NSAIDs) were inhibitors of this transport process.101 This could mean that NSAIDs have a dual anti-inflammatory activity in terms of their ability to inhibit both the synthesis and release of prostaglandins from cells. Cyclic nucleotides, signalling molecules that control cell migration, are also endogenous substrates of MRP4. Thus, MRP4 may play a role in the regulation of intracellular cyclic nucleotide levels and subsequently affect the migration of fibroblasts, which could have significance in wound repair.102 Xenobiotic substrates for MRP4 are numerous and include the antibiotics ceftizoxime and cefazolin, and the immunosuppressant methotrexate.98 The expression of MRP4 at the BBB has been shown to be localised to the luminal membrane by immunofluorescence.103–105 Quantitative mass spectrometry studies have also detected MRP4 protein in human brain endothelial cells.89 In agreement with these results, ABCC4 mRNA was also detectable in two other studies utilising isolated human brain microvessels.106,107 The Mrp4 / knockout mouse was first utilised in 2004 to show the enhanced accumulation of the anticancer agent topotecan in brain tissue and CSF.104 It was proposed that the presence of MRP4 in the BBB can confer resistance to topotecan and protect the brain from this chemotherapeutic drug.104 In a mouse brain endothelial cell line (bEnd.30), MRP4 transport was assessed by utilising siRNA targeting, and inhibition of MRP4 was shown to increase the uptake of its substrate azidothymidine Drug Transporters at the Blood–Brain Barrier 165 (an antiretroviral drug) into the brain, suggesting the functional importance of MRP4 in this in vitro model of the BBB.108 Interplay between MRP4 and other efflux transporters at the BBB can occur and is discussed in Section 5.3.5. 5.3.4 Putatively Expressed BBB Efflux Transporters In addition to MRP4, other MRP transporters have been linked to the BBB. One of these is MRP1 (ABCC1). MRP1 contains three TMDs and two NBDs. This five domain structure and the cytoplasmic N-terminus of MRP1 is atypical of ABC proteins, which generally have two TMDs and two NBDs.99 Proteomic analysis of isolated human brain microvessels failed to detect the protein above the limit of quantification, but an immunolocalisation study reported weak BBB expression in the abluminal (basolateral) membrane.89,105 However, another study detected MRP1 at the luminal membrane of brain endothelial cells.103 In addition, other studies also confirmed mRNA expression in isolated human brain microvessels.106,107 However, MRP1 does not appear to have functional activity at the BBB when tested in vivo.109 These negative experiments included work in Mdr1a / knockout mice treated with chemical inhibitors of MRP1 (probenecid or MK571) with etoposide as a substrate, or alternatively etoposide, vincristine and doxorubicin as substrates in Mrp1 / knockout mice.109 The MRP5 (ABCC5) transporter has also been linked to the BBB. MRP5, like MRP4, is a short MRP containing two TMDs and NBDs. MRP5 is expressed in many human tissues with mRNA expression in the brain and much lower mRNA levels detectable in the liver.110 No protein expression was found above the detection limit by quantitative mass spectrometry in isolated human brain microvessels,89 but two independent immunolocalisation studies revealed MRP5 protein expression on the luminal (apical) side of brain endothelial cells.103,105 In addition, MRP5 mRNA has been detected in isolated human brain microvessels.106,107 The substrate profile appears to have a distinct overlap with MRP4, particularly with regards to nucleotides.110 However, its functional role in the BBB for transport of xenobiotics has yet to be fully determined. 5.3.5 Interplay Between Efflux Transporters The efflux transporters expressed at the BBB can work in tandem to exclude a drug from the brain93 because of their overlapping substrate specificity. In practice, this means that if one transporter is knocked out or inhibited, then the other efflux transporter(s) may compensate by excluding the compound from the brain. Numerous examples of drugs that are substrates for dual efflux transporters at the BBB now exist, including a number of tyrosine kinase inhibiters used in cancer treatment. At the mouse BBB, imatinib entry into the brain has been found to be restricted not only by P-gp (see Section 5.3.1) but also by BCRP.78,111 Using 166 Chapter 5 / knockout mice, the brain concentration of imatinib in Abcg2 mice was similar to that in wild type mice, whereas the concentration in Mdr1a/1b / and Mdr1a/1b / Bcrp / mice was increased more than 3- to 4-fold and 19to 50-fold, respectively, compared with wild type mice. This clearly shows the synergistic activity of these transporters at the BBB for imatinib transport. Another example is the drug sunitinib, which has been approved by the US Food and Drug Administration (FDA) for the treatment of renal cell carcinoma and imatinib resistant gastrointestinal stromal tumour. The brain accumulation of sunitinib is restricted by P-gp and BCRP, and can be enhanced by oral elacridar co-administration in knockout mice studies.112 The oral availability and brain penetration of the B-RAF mutant inhibitor (V600E) vemurafenib are affected by efflux transporters at the BBB. They can be enhanced by inhibition of both P-gp and BCRP by elacridar treatment, resulting in increased brain accumulation in mice.113 While studies in knockout mice found that the brain to plasma ratios of vemurafenib were only increased 1.7-fold in Mdr1a/1b / mice, and not increased in Abcg2 / mice, ratios were increased by 21-fold in triple knockout Mdr1a/1b / Abcg2 / mice, clearly showing the importance of both of these transporters in vemurafenib brain penetration. Similarly, URB937, a fatty acid amide hydrolase inhibitor (FAAH), is a dual substrate for P-gp and BCRP, and these efflux transporters restrict its access to the brain.114 Thus, URB937 is restricted to the periphery, revealing an unexpected role for fatty acid amide hydrolase in pain initiation control outside of the CNS. This phenomenon of interplay between efflux transporters at the BBB is not just limited to BCRP and P-gp. An example of this is methotrexate, with an effect on brain penetration only being observed when both BCRP and MRP4 are knocked out in rodents.115 It is also possible for a drug to be a substrate of all three of the main efflux transporters expressed at the BBB, i.e. P-gp, MRP4 and BCRP. An example of this is the antitumour camptothecin analogues. Investigations using knockout mice found that these drugs are restricted by MRP4 together with P-gp and BCRP, thus forming a robust cooperative drug efflux system that restricts their entry into the brain.116 This cooperative drug efflux system is also exemplified by lapatinib, a dual tyrosine kinase inhibitor. Whilst the double knockout Mdr1a/b / mice only showed a 3- to 4-fold increase in brain concentration and Abcg2 / mice had no increase when compared with wild type mice, the Mdr1a/b / Abcg2 / triple knockout animal was found to have a 40-fold increase in brain concentration compared with the wild type mice.117 This type of work has led to the suggestion that inhibitors of efflux transporters could be used to boost the brain penetration of drugs through inhibition of efflux transporters at the BBB; however, even if suitable inhibitors could be found, a drawback of this approach is that this could lead to toxic side effects in other organs expressing the transporter(s) of interest (see Section 5.6). Drug Transporters at the Blood–Brain Barrier 167 5.4 Influx Transporters Expressed at the BBB Secondary-active and facilitative transporters belong to the superfamily of solute carrier (SLC) transporters. SLCs represent the largest known superfamily of membrane transporters and the official list currently consists of over 300 SLC transporter encoding genes within 52 families (SLC1–SLC52) and is available from the Human Genome Organisation Gene Nomenclature Committee at the European Bioinformatics Institute (HGNC) website (http:// slc.bioparadigms.org/). A corresponding review article for each family has recently been published in Molecular Aspects of Medicine118 and some selected families that have relevance to the BBB are highlighted in more detail in this chapter. SLC genes are allocated to a subfamily if the coded proteins share at least 20% of the amino acid sequence, and can be divided into further subfamilies.118 The official nomenclature for each gene starts with SLC to indicate the corresponding gene superfamily and is followed by the family number and subfamily letter. Finally, each gene is allocated a unique number. The SLC superfamily comprises a diverse set of transporters involved in the absorption and excretion of a broad spectrum of physiologically important endogenous molecules/ions, but also xenobiotics such as drugs. For example, members of the SLC2A family are essential for an adequate sugar supply to the brain and other organs, while the SLC39A family is essential for metal ion homeostasis, particularly zinc.119,120 However, uncertainty exists around which influx transporters are important at the BBB. This is therefore a developing area of research, with a recent and major review on SLC transporters calling for systematic research on this important but relatively uncharacterised family of proteins.121 The current knowledge of the likely expression of certain transporters is summarised in Figure 5.5. 5.4.1 LAT1 LAT1 (SLC7A5) was cloned at the end of the 1990s and found to be highly expressed in brain endothelial cells.106,122,123 It is an amino acid antiporter that pumps a substrate in and then effluxes an internal substrate out of the cell with a 1 : 1 stoichiometry.124 The amino acids that LAT1 transports are neutral amino acids such as phenylalanine and leucine.125 LAT1 forms a heterodimer with CD98 (SLC3A2, 4F2 heavy chain), which is proposed to aid in the localisation of LAT1 to the plasma membrane. As well as being a nutrient transporter, a number of drugs have been shown to be substrates for LAT1. These include L-DOPA and gabapentin.37,126 The knockout mouse for LAT1 is embryonic lethal, which has restricted research on this transporter. However, the recent use of a conditional knockout in T-cells has, at least for T-cell differentiation, shown an essential requirement for LAT1.127 At the BBB, LAT1 is thought to be localised to both the luminal and abluminal membranes of brain endothelial cells, as demonstrated by immunofluorescence studies.128,129 The use 168 Chapter 5 Figure 5.5 Subcellular expression of influx transporters at the BBB. SLC transporters at the BBB that have been introduced in this chapter with confirmed expression are LAT1 (SLC7A5) and GLUT1 (SLC2A1) (in purple). The putative expression of OATP1A2 (SLCO1A2) and OATP2B1 (SLCO2B1) is shown in yellow with their proposed subcellular expression; other SLC transporters have also been proposed (see text). of either competitive inhibitors or siRNA targeted to LAT1 has shown its functional importance in brain endothelial cells of mouse and human origin.37,130 Due to LAT1 enrichment and functional expression at the BBB, LAT1 is considered a promising target for drug delivery to the brain (see Section 5.7). 5.4.2 Organic Anion Transporting Polypeptide Transporters The organic anion transporting polypeptide transporters (OATPs) are a class of influx transporters that are part of the SLC superfamily and, more specifically, encoded by the solute carrier organic anion (SLCO) gene subfamily. The human OATP family consists of 11 members. Controversy surrounds which, if any of the OATPs are functionally important at the BBB.131 However, this class of transporter has been shown to have an important role in the influx of organic anions at other tissue types such as the kidney and liver.131 OATP1A2 (OATP-A, SLCO1A2) was the first OATP to be linked to expression in human brain endothelial cells.132 Since this finding, OATP2B1 (SLCO2B1) has also been suggested to be expressed in human brain endothelial cells in paraffin embedded sections.133 However, in quantitative mass spectrometry studies no OATP protein was detected above the limits of detection in brain endothelial cells.89 Rodent OATP1A4 (Slco1a4) has been suggested to function at the BBB, with its transport function being Drug Transporters at the Blood–Brain Barrier 169 upregulated in hypoxia, leading to more drug being delivered to the brain.134,135 The human counterpart to rodent OATP1A4 is unclear, so the relevance of this finding for the human BBB is unknown; however it does share 72% sequence homology at the amino acid level to human OATP1A2.134 Further research is required to determine which, if any, OATP is functionally important at the human BBB. 5.4.3 Monocarboxylate Transporters Monocarboxylic acid transporters (MCTs; SLC16A) comprise a family of at least eight proton-coupled transporters.136 MCT1 can transport endogenous substrates such as lactate and drug substrates such as salicylate, atorvastatin, nateglinide, g-hydroxybutyrate and nicotinic acid.136 A number of in vivo experiments in rodents and in vitro studies with brain endothelial cells suggest that the BBB has a monocarboxylate transport system.136 MCT1 protein expression has been detected in the brain endothelial cells of the BBB with the assumption, due to a lack of further evidence, that this is the main MCT expressed at the BBB.137 The significance of MCT-mediated transport of drug substrates at the BBB is at present unknown, but a recent study found that MCT1 was downregulated in brain microvessels in patients with temporal lobe epilepsy.138 5.4.4 Organic Cation Transporters Organic cation transporters (OCTs) comprise a class of transporters that are part of the SLC22 subfamily. OCT1 (SLC22A1), OCT2 (SLC22A2) and OCT3 (SLC22A3) constitute the first subgroup of functionally characterised transporters from the SLC22A family. Corresponding to their name, OCTs predominantly recognise organic cations or weakly alkaline molecules that are positively charged at physiological pH.139 Around 40% of all orally administered drugs exhibit these physicochemical properties140 and, accordingly, a large number of compounds have been found to interact with OCT1–3, particularly as inhibitors, with broadly overlapping but distinctive specificities.139 The translocation of substrates follows a facilitated, bi-directional mechanism down an electrochemical gradient.139 Drug library screening for OCT1 and OCT2 inhibition has revealed some common physicochemical features of potent inhibitors, including a positive net charge and high lipophilicity.141 At the mRNA level, OCT1 is most abundantly expressed in the liver but is also detectable in the intestine, kidney, brain and other organs. The protein is localised to the luminal (apical) membrane of brain endothelial cells,142–145 which could result in OCT1 mediating the uptake of substrates from the blood into the brain. OCT2 mRNA is most significantly detectable in the kidney but also present in the brain, intestine and other tissues.139 The protein is localised in the luminal (apical) membrane of brain endothelial cells.144,146 OCT3 transcripts are detectable in various tissues 170 Chapter 5 139 including the liver, kidney, brain and intestine. Protein expression of OCT3 has recently been shown in isolated human brain microvessels, although its subcellular localisation remains to be determined.106 The role of specific organic cation transporter(s) in BBB transport is unclear. However, studies have linked different OCTs to the transport of a number of drugs at the BBB. These include the neurotoxin MPTP, lamotrigine, amisulpride and sulpiride.36,144,147,148 Either multiple different transporters are important for the uptake of these drugs or an uncharacterised BBB specific organic cation-like transporter has functional importance at the BBB. For example, both choline and pyrilamine have been linked to a carriage process mediated by an unidentified cation transporter at the BBB, termed either the pyrilamine or choline transporter.149,150 5.4.5 Organic Anion Transporters The organic anion transporters (OATs), OAT1–3, OAT7, OAT4, URAT1 and OAT10 proteins (SLC22A6–9 and SLC22A11–13) belong to the third and largest subgroup of membrane transporters from the SLC22A family. They mediate the transport of organic anions in either direction and are particularly important in the first step of renal excretion.151 Most, if not all, are secondary-active organic anion exchangers utilising counter-ions such as a-ketoglutarate, lactate and nicotinate.151 To date, it remains unclear whether any of the OATs/URAT1 are involved in human BBB transport.152 A quantitative absolute proteomics study with isolated human brain microvessels attempted to analyse the expression of 114 transporters at the human BBB.89 None of the investigated organic anion uptake transporters were above the limit of quantification. However, the same negative results were obtained in this study for the entire SLC22A family and all 11 human SLCO (OATP) transporters.89 Caution in the interpretation of these results is therefore warranted, as there may be issues with the sensitivity of the method used, although other transporters of the ABC family, such as P-gp, BCRP and MRP4, were detected. In contrast, recent work that analysed the mRNA expression of most SLC transporters in isolated human brain microvessels found that the majority of the SLC22A genes were expressed.106 At least in rodents, a candidate OAT at the BBB is OAT3. OAT3 has been found to be localised to the abluminal membrane in brain endothelial cells of mouse and rat origin.153,154 In one study, an active metabolite of oseltamivir (Ro 64-0802), used in the treatment of influenza virus, was microinjected into the cerebrum of knockout Oat3 / mice. The amount of Ro 64-0802 in the brain was significantly greater in the Oat3 / knockout mice than wild type mice 2 hours after the microinjection, suggesting that OAT3 is a candidate transporter for this compound at the BBB.155 An additional study utilised a model organic anion drug (dehydroepiandrosterone sulfate) as a probe substrate of OAT3 and investigated its efflux from the mouse brain. The elimination of the probe compound from the brain after microinjection into the cerebral cortex was found to be delayed in Oat3 / Drug Transporters at the Blood–Brain Barrier 171 knockout mice compared with the controls, suggesting a functional role of OAT3 at the mouse BBB.156 How this in vivo work in mice correlates to human BBB transport is, however, as yet unknown. 5.4.6 Nutrient Transporters As the BBB is involved in maintaining brain homeostasis, a number of influx transporters that transport endogenous nutrients are expressed. These include LAT1 (see Section 5.4.1), GLUT1 and MFSD2A. GLUT1 (SLC2A1) is part of the sugar porter subfamily of the major facilitator superfamily (MFS), one of the largest secondary transporter superfamilies. GLUT1 is highly expressed at the BBB with subcellular localisation at both the luminal and abluminal membranes of brain endothelial cells, and has the essential role of transporting glucose into the brain.89,157–159 Mutations of GLUT1 that affect activity can result in reduced transport of glucose and are associated with neurological syndromes as a result of lack of energy supply to the brain.160 The crystal structure of the human glucose transporter GLUT1 has been solved recently and this gives insight into its mode of action.161 GLUT1 has no known drug substrates but interactions with drugs have been observed, for example barbiturates and sodium valproate in vitro can reduce GLUT1-mediated transport of glucose.162,163 However, how or if this could affect the role of the BBB in maintaining brain homeostasis is still unknown. MFSD2A is a novel omega three fatty acid transporter that was recently identified at the mouse BBB,13,164 and is thought to be a key regulator of BBB function. MFSD2A has no known drug substrates but it has recently been shown to be a putative transporter of tunicamycin, which is used as an in vitro chemical tool to induce the unfolded protein response.165 The recent identification of MFSD2A at the BBB is a good example of how additional influx transporters are being found and how this is an ongoing area of study. 5.5 Transporters Expressed at the CP CP epithelial cells are the main site of CSF secretion and, along with the arachnoid membrane, form the blood–CSF barrier. In brief, the CP is a vascularised tissue that is located in each ventricle of the brain. The CP epithelial cells form a monolayer of cells that are joined together by tight junctions to form the blood–CSF barrier, which prevents the paracellular passage of molecules from the circulatory system into the CSF.166 Like BBB endothelial cells, the CP epithelial cells express a variety of transporters. These transporters are involved in maintaining CSF homeostasis such as movement of nutrients or waste products out/into the CSF, and the transport of pharmaceutical drugs in/out of the CSF.167 The concentration of a drug in the CSF is considered to be the product of transport through the CP epithelium cells and as such does not readily correlate with brain concentrations of a compound.65 A limited number of gene deletion studies using knockout mice have been used to investigate the significance of transporters 172 Chapter 5 167 at the rodent CP, as outlined in an extensive review. This review details immunohistochemical studies of transporter localisation in knockout mice that have determined a major difference between drug efflux transporter proteins in CP epithelial cells compared with those in the brain endothelial cells of the BBB. P-gp and BCRP in the CP epithelial cells were found to be located on the CSF-facing membrane, while at the BBB these transporters were localised at the blood-facing membrane.167 This results in the P-gp and BCRP transporters at the BBB pumping substrates out of the brain, while in the CP they pump substrates into the CSF. Such a differential effect of the transporters at the two CNS barriers emphasises the difference in the basic biology and explains, at least in part, why the CSF is not always a good surrogate for the brain concentration of a drug. 5.6 Challenge Up to 98% of all drugs may fail to cross the BBB at pharmacologically relevant concentrations, which has resulted in a major challenge for the development of CNS drugs to combat neurological disorders and brain tumours.28 The traditional method has been to use medicinal chemistry approaches to alter the properties of a potential candidate compound in order to enhance its lipid solubility, but this approach has failed to produce many effective treatments for CNS diseases. This is due, at least in part, to efflux transporters such as P-gp transporting lipophilic substrate compounds out of the brain (see Section 5.3). Alternatively, if a drug is transported by an influx transporter, this could cause issues such as carriage across the BBB and undesired CNS side effects. One example of a CNS adverse effect is with tecadenoson (CVT-510), an A1 adenosine receptor agonist that has undergone small scale clinical trials due to its potent anti-arrhythmic effects in tachycardias.168 A1 adenosine receptors are expressed both on cardiac and brain tissues, and the drug is known to cause CNS sedative effects in humans.168,169 A recent study has shown that tecadenoson is a substrate of nucleoside transporters in vitro and in vivo in mice, with inhibition of the equilibrative nucleoside transporter 1 (ENT1) reducing the BBB transport of the drug.169 Cheminformatics approaches have recently suggested that pharmaceutical drugs that have a structural similarity to cellular metabolites and nutrients can have a similar transport profile to their homologous cellular metabolite.170,171 A recent example of how this concept of metabolite or nutrient likeness may be important, but is sometimes overlooked, is with fedratinib. Fedratinib is a Janus kinase 2 inhibitor that was in a Phase III trial for the treatment of myelofibrosis, but was withdrawn due to the onset of Wernicke’s-like encephalopathy in a subset of patients.172 Fedratinib has structural similarities to thiamine and was subsequently found to interact directly with the thiamine transporter [SLC19A3, thiamine transporter 2 (THTR2)], providing a clear explanation for the onset of Wernicke’s encephalopathy and an example of a drug–nutrient interaction at the 173 Drug Transporters at the Blood–Brain Barrier 173 transporter level with a serious adverse reaction. This study utilised Caco-2 cells as an oral absorption model but also found that the free drug concentration of fedratinib in the rodent brain was higher than expected compared with its physicochemical properties. A study from the 1980s showed that thiamine at the BBB has a saturable active transport component.174 If this transporter-mediated carriage was also the case for fedratinib at the BBB, this could then be an explanation of why this compound was able to penetrate the brain at levels higher than predicted. It is also known that certain diseases may affect the expression of transporters at the BBB and the tightness of the barrier. For example in Alzheimer’s disease, a PET study in humans has shown decreased activity of P-gp at the BBB,175 and animal models of peripheral pain have indicated increased P-gp expression at the rodent BBB, which reduces morphine penetration into the brain.176 HIV infection of a limited number of astrocytes at the BBB is known to result in a disrupted BBB and is proposed to be a cause of the HIV-associated cognitive impairment that can occur in infected individuals.177 The relevance of this affect for antiretroviral drug uptake into this sanctuary site is unknown. In addition, transporter activity at the BBB can be affected by diseases or impairment in other organs in the body, for example liver damage may lead to increased P-gp and MRP2 expression and function at the BBB in rodents.178 Ongoing research will no doubt shed further light on the changes at the BBB during disease processes and how they might affect drug delivery via changes in drug transporters. 5.7 Opportunity As well as challenges, opportunities for targeting drugs to the CNS also exist due to the expression of drug transporters at the BBB. This includes strategies to overcome the lack of brain penetration by targeting the mechanism of uptake; targeting efflux transporter(s) at the BBB so that a drug is excluded from the brain, thus potentially avoiding CNS side effects; and using transport inhibitors to boost the uptake of a drug into the brain. An interaction of a drug with an efflux transporter is not necessarily a negative outcome, since this can result in a drug being excluded from the brain and avoiding potentially serious CNS side effects. An example of this is with sedating and non-sedating antihistamines. Cetirizine and hydroxyzine are related structurally, but cetirizine has a non-sedating profile, due to the fact that it is a P-gp substrate and is excluded from the CNS, whereas hydroxyzine is not a P-gp substrate and can be used as a sedative drug.179 The use of efflux transporter inhibitors has been proposed as an adjuvant treatment to target drugs to the brain that would otherwise be excluded by P-gp and/or BCRP, with success being achieved in preclinical animal studies. Although this could be of particular relevance to, for example, tyrosine kinase inhibitors, for which both P-gp and BCRP at the BBB have been linked to the lack of brain uptake that restricts their applications in the treatment of brain tumours, this approach has failed to translate into the 174 Chapter 5 clinic. Early generation transport inhibitors have failed in clinical trials as adjuvants for cancer therapy, due to either a lack of clinical benefit or high incidence of toxicity,180 and no P-gp inhibitor has yet received approval from the European regulatory agencies or the US FDA as an adjuvant treatment.180,181 The situation with third generation inhibitors such as tariquidar or elacridar showed promise in early phase clinical trials,182,183 but in the case of tariquidar, a Phase III trial in non-small-cell lung cancer was terminated due to safety concerns. An area of basic research that is in its infancy is the modulation of drug transporter expression to achieve more drug penetration to the target site, which could have great potential in the long term.184 Targeting influx transporters expressed at the BBB to achieve uptake of the drug into the brain is an opportunity that is attracting current interest. Historically this has happened more by accident than design, a classic example being L-DOPA. Dopamine does not cross the BBB but a prodrug of dopamine, L-DOPA, has high permeability across the BBB and is a substrate of LAT1, a BBB influx transporter.123,126,185 Subsequently, a number of groups developed compounds that are transported by LAT1, enabling delivery to the brain; these are at an early stage but appear promising.186,187 Utilising in silico modelling, Geier et al. were able to predict and experimentally validate four novel ligands of LAT1.188 With the advance of structural biology in terms of solving the structure of a number of SLC transporters, this opens up the realistic possibility of solving the structure of BBB influx transporters and thus enabling QSAR to be performed to specifically target ligands to the BBB transporter and thus the brain.161,189 Drawbacks to this methodology are not yet clear but could include drug– nutrient interactions and saturation by the drug of the transporter-mediated process. 5.8 Summary In this chapter we have outlined the physiology of the BBB and how drug transporters are an important consideration in how drugs are able to penetrate into the brain. 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