Barrière hématoencéphalique Partie III : approche thérapeutique pour franchir la barrière hématoencéphalique

Over the last few years, the blood-brain barrier has come to be considered as the main limitation for the treatment of neurological diseases caused by inflammatory, tumor or neurodegenerative disorders. In the blood-brain barrier, the close intercellular contact between cerebral endothelial cells due to tight junctions prevents the passive diffusion of hydrophilic components from the bloodstream into the brain. Several specific transport systems (via transporters expressed on cerebral endothelial cells) are implicated in the delivery of nutriments, ions and vitamins to the brain; other transporters expressed on cerebral endothelial cells extrude endogenous substances or xenobiotics, which have crossed the cerebral endothelium, out of the brain and into the bloodstream. Recently, several strategies have been proposed to target the brain, (i) by by-passing the blood-brain barrier by central drug administration, (ii) by increasing permeability of the blood-brain barrier, (iii) by modulating the expression and/or the activity of efflux transporters, (iv) by using the physiological receptor-dependent blood-brain barrier transport, and (v) by creating new viral or chemical vectors to cross the blood-brain barrier. This review focuses on the illustration of these different approaches.     

Brain-to-blood transporters for endogenous substrates and xenobiotics at the blood-brain barrier: An overview of biology and methodology

In the past decade, research into P-glycoprotein involving the blood-brain barrier (BBB) has seen a shift in the concept of the BBB as a structural barrier to that of a functional barrier for xenobiotics and changed simultaneously the strategy for the discovery and development of drugs acting in the CNS. As far as making advances in neurotherapeutics are concerned, the brain-to-blood transport function at the BBB will be one of the most important issues. Knowing the limitations of thein vivo andin vitro methods for BBB efflux research, it is essential to adopt a multidisciplinary approach in investigating the true physiological role of the BBB. Among several methods, the Brain Efflux Index method and the use of conditionally immortalized brain capillary endothelial cell lines, established from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene, are likely to be very useful tools for the BBB efflux transport research. According to our recent findings using these methods, several transporters in the brain capillary endothelial cells appear to play an important role in reducing the brain level of hydrophilic endogenous substrates produced either in the brain or peripheral organs, e.g., neurotransmitters, neuromodulators, metabolites of neurotransmitters, and uremic toxins. It has been reported also that large hydrophilic molecules, such as IgG, apo-transferrin, and amyloid-β peptide, are susceptible to brain-to-blood efflux transport. In the light of the latest findings, we have formed the hypothesis that the BBB acts as a CNS detoxifying system for both endogenous substrates and xenobiotics in the brain. A fuller understanding of the physiological role of BBB efflux transporters will provide rational insights to assist in the development of safer neurotherapeutics.     

Membrane transporter proteins: a challenge for CNS drug development

Drug transporters are membrane proteins present in various tissues such as the lymphocytes, intestine, liver, kidney, testis, placenta, and central nervous system. These transporters play a significant role in drug absorption and distribution to organic systems, particularly if the organs are protected by blood-organ barriers, such as the blood-brain barrier or the maternal-fetal barrier. In contrast to neurotransmitters and receptor-coupled transporters or other modes of interneuronal transmission, drug transporters are not directly involved in specific neuronal functions, but provide global protection to the central nervous system. The lack of capillary fenestration, the low pinocytic activity and the tight junctions between brain capillary and choroid plexus endothelial cells represent further gatekeepers limiting the entrance of endogenous and exogenous compounds into the central nervous system. Drug transport is a result of the concerted action of efflux and influx pumps (transporters) located both in the basolateral and apical membranes of brain capillary and choroid plexus endothelial cells. By regulating efflux and influx of endogenous or exogenous substances, the blood-brain barrier and, to a lesser extent the blood-cerebrospinal barrier in the ventricles, represents the main interface between the central nervous system and the blood, i.e., the rest of the body. As drug distribution to organs is dependent on the affinity of a substrate for a specific transport system, membrane transporter proteins are increasingly recognized as a key determinant of drug disposition. Many drug transporters are members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily or the solute-linked carrier (SLC) class. The multidrug resistance protein MDR1 (ABCB1), also called P-glycoprotein, the multidrug resistance-associated proteins MRP1 (ABCC1) and MRP2 (ABCC2), and the breast cancer-resistance protein BCRP (ABCG2) are ATP-dependent efflux transporters expressed in the blood-brain barrier They belong to the superfamily of ABC transporters, which export drugs from the intracellular to the extracellular milieu. Members of the SLC class of solute carriers include, for example, organic ion transporting peptides, organic cation transporters, and organic ion transporters. They are ATP-independent polypeptides principally expressed at the basolateral membrane of brain capillary and choroid plexus endothelial cells that also mediate drug transport through central nervous system barriers.     

Coordinated nuclear receptor regulation of the efflux transporter, Mrp2, and the phase-II metabolizing enzyme, GSTpi, at the blood-brain barrier.

Xenobiotic efflux pumps at the blood-brain barrier are critical modulators of central nervous system pharmacotherapy. We previously found expression of the ligand-activated nuclear receptor, pregnane X receptor (PXR), in rat brain capillaries, and showed increased expression and transport activity of the drug efflux transporter, P-glycoprotein, in capillaries exposed to PXR ligands (pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone) in vitro and in vivo. Here, we show increased protein expression and transport activity of another efflux pump, multidrug resistance-associated protein isoform 2 (Mrp2), in rat brain capillaries after in vitro and in vivo exposure to PCN and dexamethasone. The phase-II drug-metabolizing enzyme, glutathione S-transferase-pi (GSTpi), was found to be expressed in brain capillaries, where it colocalized to a large extent with Mrp2 at the endothelial cell luminal plasma membrane. Like Mrp2, GSTpi protein expression increased with PXR activation. Colocalization and coordinated upregulation suggest functional coupling of the metabolizing enzyme and efflux transporter. These findings indicate that, as in hepatocytes, brain capillaries possess a regulatory network consisting of nuclear receptors, metabolizing enzymes, and efflux transporters, which modulate blood-brain barrier function.     

Regulation and role of organic anion-transporting polypeptides (OATPs) in drug delivery at the choroid plexus

The organic anion-transporting polypeptides (rodents: Oatps; human: OATPs) belong to the growing family of organic anion/prostaglandin transporters and are important components of the active efflux transport system at the choroid plexus epithelial cells. OATPs facilitate the elimination of xenobiotics and endogenous waste from the cerebrospinal fluid and prevent waste accumulation in the central nervous system (CNS). This review summarizes the structures, regulations and roles of Oatps/OATPs at the choroid plexus in drug delivery to the CNS.     

Probenecid-inhibitable efflux transport of valproic acid in the brain parenchymal cells of rabbits: a microdialysis study

Delivery of valproic acid (VPA) to the human brain is relatively inefficient as reflected by a low brain-to-unbound plasma concentration ratio (< or =0.5) at steady state. Previous pharmacokinetic studies suggested that the unfavorable brain-to-plasma gradient is maintained by coupled efflux transport processes at both the brain parenchymal cells and blood-brain barrier (BBB); one or both of the efflux transporters are inhibitable by probenecid. The present study in rabbits utilized microdialysis to measure drug concentration in the brain extracellular fluid (ECF) of the cerebral cortex during steady-state i.v. infusion with VPA alone or with VPA plus probenecid. Probenecid co-infusion elevated VPA concentration in the brain tissue surrounding the tip of the microdialysis probe to a greater extent than in the ECF (230% versus 47%). Brain intracellular compartment (ICC) concentration was estimated. In control rabbits, the ICC concentration was 2.8+/-0.28 times higher than the ECF concentration. Probenecid co-infusion elevated the ICC-to-ECF concentration ratio to 4.2+/-0.44, which confirms the existence of an efflux transport system in brain parenchymal cells. The ECF-to-unbound plasma concentration ratio was well below unity (0.029), indicating an uphill efflux transport of VPA across the BBB. Co-infusion of probenecid did not have a significant effect on VPA efflux at the BBB as evidenced by a minimal change in the ECF-to-unbound plasma concentration ratio. This study suggests the presence of distinctly different organic anion transporters for the efflux of VPA at the parenchymal cells and capillary endothelium in the brain.     

Blood-brain barrier active efflux transporters: ATP-binding cassette gene family

The blood-brain barrier (BBB) contributes to brain homeostasis by protecting the brain from potentially harmful endogenous and exogenous substances. BBB active drug efflux transporters of the ATP-binding cassette (ABC) gene family are increasingly recognized as important determinants of drug distribution to, and elimination from, the CNS. The ABC efflux transporter P-glycoprotein (Pgp) has been demonstrated as a key element of the BBB that can actively transport a huge variety of lipophilic drugs out of the brain capillary endothelial cells that form the BBB. In addition to Pgp, other ABC efflux transporters such as members of the multidrug resistance protein (MRP) family and breast cancer resistance protein (BCRP) seem to contribute to BBB function. Consequences of ABC efflux transporters in the BBB include minimizing or avoiding neurotoxic adverse effects of drugs that otherwise would penetrate into the brain. However, ABC efflux transporters may also limit the central distribution of drugs that are beneficial to treat CNS diseases. Furthermore, neurological disorders such as epilepsy may be associated with overexpression of ABC efflux transporters at the BBB, resulting in pharmacoresistance to therapeutic medication. Therefore, modulation of ABC efflux transporters at the BBB forms a novel strategy to enhance the penetration of drugs into the brain and may yield new therapeutic options for drug-resistant CNS diseases.     

Mechanisms of glutamate efflux at the blood-brain barrier: involvement of glial cells

At high concentrations, glutamate (Glu) exerts potent neurotoxic properties, leading to irreversible brain damages found in numerous neurological disorders. The accepted notion that Glu homeostasis in brain interstitial fluid is maintained primarily through the activity of Glu transporters present on glial cells does not take into account the possible contribution of endothelial cells constituting the blood-brain barrier (BBB) to this process. Here, we present evidence for the presence of the Glu transporters, excitatory amino-acid transporters (EAATs) 1 to 3, in porcine brain endothelial cells (PBECs) and show their participation in Glu uptake into PBECs. Moreover, transport of Glu across three in vitro models of the BBB is investigated for the first time, and evidence for Glu transport across the BBB in both directions is presented. Our results provide evidence that the BBB can function in the efflux mode to selectively remove Glu, via specific transporters, from the abluminal side (brain) into the luminal compartment (blood). Furthermore, we found that glial cells lining the BBB have an active role in the efflux process by taking up Glu and releasing it, through hemichannels, anion channels, and possibly the reversal of its EAATs, in close proximity to ECs, which in turn take up Glu and release it to the blood.     

Small molecular drug transfer across the blood-brain barrier via carrier-mediated transport systems

Because of the physiological nature of the blood-brain barrier (BBB), transport of chemical compounds between blood and brain has been widely believed to occur by means of passive diffusion, depending upon the lipophilicity of the compounds. However, discrepancies exist between the lipophilicity and apparent BBB permeation properties in many cases, and these discrepancies can be ascribed to the existence of multiple mechanisms of drug transport through the BBB. Molecular identification and functional analysis of influx transport proteins (from blood to brain) and efflux transport proteins (from brain to blood) have progressed rapidly. Therefore, the BBB is now considered to be a dynamic interface that controls the influx and efflux of a wide variety of substances, including endogenous nutrients and exogenous compounds such as drugs, to maintain a favorable environment for the CNS. This review focuses on the role of transport systems in the uptake of xenobiotics, including organic anionic/cationic and neutral drugs, across the BBB into the brain, as well as on strategies to increase drug delivery into the brain by blocking efflux transport protein function, or to reduce CNS side effects by modulating BBB transport processes.     

Glucose transporter proteins in brain: Delivery of glucose to neurons and glia

Glucose is the principle energy source for the mammalian brain. Delivery of glucose from the blood to the brain requires transport across the endothelial cells of the blood-brain barrier and into the neurons and glia. The facilitative glucose transporter proteins mediate these processes. The primary isoforms in brain are GLUT1, detected at high concentrations as a highly glycosylated form, (55 kDa) in blood-brain barrier, and also as a less glycosylated, 45 kDa form, present in parenchyma, predominantly glia; GLUT3 in neurons; and GLUT5 in microglia. The rest of the transporter family, GLUTs 2, 4, and 7, have also been detected in brain but at lower levels of expression and confined to more discrete regions. All of the transporters probably contribute to cerebral glucose utilization, as part of overall metabolism and metabolic interactions among cells. We discuss the properties, regulation, cell-specific location, and kinetic characteristics of the isoforms, their potential contributions to cerebral metabolism, and several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. GLIA 21:2–21, 1997. © 1997 Wiley-Liss, Inc.     

Blood-brain barrier flux of aluminum, manganese, iron and other metals suspected to contribute to metal-induced neurodegeneration

The etiology of many neurodegenerative diseases has been only partly attributed to acquired traits, suggesting environmental factors may also contribute. Metal dyshomeostasis causes or has been implicated in many neurodegenerative diseases. Metal flux across the blood-brain barrier (the primary route of brain metal uptake) and the choroid plexuses as well as sensory nerve metal uptake from the nasal cavity are reviewed. Transporters that have been described at the blood-brain barrier are listed to illustrate the extensive possibilities for moving substances into and out of the brain. The controversial role of aluminum in Alzheimer's disease, evidence suggesting brain aluminum uptake by transferrin-receptor mediated endocytosis and of aluminum citrate by system Xc;{-} and an organic anion transporter, and results suggesting transporter-mediated aluminum brain efflux are reviewed. The ability of manganese to produce a parkinsonism-like syndrome, evidence suggesting manganese uptake by transferrin- and non-transferrin-dependent mechanisms which may include store-operated calcium channels, and the lack of transporter-mediated manganese brain efflux, are discussed. The evidence for transferrin-dependent and independent mechanisms of brain iron uptake is presented. The copper transporters, ATP7A and ATP7B, and their roles in Menkes and Wilson's diseases, are summarized. Brain zinc uptake is facilitated by L- and D-histidine, but a transporter, if involved, has not been identified. Brain lead uptake may involve a non-energy-dependent process, store-operated calcium channels, and/or an ATP-dependent calcium pump. Methyl mercury can form a complex with L-cysteine that mimics methionine, enabling its transport by the L system. The putative roles of zinc transporters, ZnT and Zip, in regulating brain zinc are discussed. Although brain uptake mechanisms for some metals have been identified, metal efflux from the brain has received little attention, preventing integration of all processes that contribute to brain metal concentrations.     

Mechanisms of glucose transport at the blood-brain barrier: an in vitro study

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood-brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood-brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood-brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood-brain barrier model of approximately 0.85. Under conditions where [glucose](lumen) was higher than [glucose](ablumen), influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.     

Xenobiotic transport and metabolism in the human brain

Organisms have metabolic pathways responsible for eliminating endogenous and exogenous toxicants. Generally, we associate the liver par excellence as the organ in charge of detoxifying the body; however, this process occurs in all tissues, including the brain. Due to the presence of the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), the Central Nervous System (CNS) is considered a partially isolated organ, but similar to other organs, the CNS possess xenobiotic transporters and metabolic pathways associated with the elimination of xenobiotic agents. In this review, we describe the different systems related to the detoxification of xenobiotics in the CNS, providing examples in which their association with neurodegenerative processes is suspected. The CNS detoxifying systems include carrier-mediated, active efflux and receptor-mediated transport, and detoxifying systems that include phase I and phase II enzymes, as well as those enzymes in charge of neutralizing compounds such as electrophilic agents, reactive oxygen species (ROS), and free radicals, which are products of the bioactivation of xenobiotics. Moreover, we discuss the differential expression of these systems in different regions of the CNS, showing the different detoxifying needs and the composition of each region in terms of the cell type, neurotransmitter content, and the accumulation of xenobiotics and/or reactive compounds.     

In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate

The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of ³H-L-glutamate, ³H-L-aspartate, and ³H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm², and ¹⁴C-D-mannitol permeability values of 0.88 ± 0.13 × 10⁻⁶ cm s⁻¹. Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K_m of 69 ± 15 μM and a J_max of 44 ± 3.1 pmol min⁻¹ cm⁻² for L-aspartate and a K_m of 138 ± 49 μM and J_max of 28 ± 3.1 pmol min⁻¹ cm⁻² for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), −2 (Slc1a2), and −3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and −3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations. © 2012 Wiley Periodicals, Inc.     

Differential axial localization along the mouse brain vascular tree of luminal sodium-dependent glutamine transporters Snat1 and Snat3

A specialized brain vasculature is key for establishing and maintaining brain interstitial fluid homeostasis, which for most amino acids (AAs) are ∼10% plasma levels. Indeed, regulation of AA homeostasis seems critical for normal central nervous system functions, and disturbances in brain levels have both direct and indirect roles in several neuropathologies. One mechanism contributing to the plasma to brain AA gradients involves polarized expression of solute carrier (SLC) family transporters on blood–brain barrier (BBB) endothelial cells. Of particular interest is the localization of sodium-dependent transporters that can actively move substrates against their concentration gradient. In this study, the in vivo endothelial membrane localization of the sodium-dependent glutamine transporters Snat3 (Slc38a3) and Snat1 (Slc38a1) was investigated in the mouse brain microvasculature using immunofluorescent colocalization with cellular markers. In addition, luminal membrane expression was probed by in vivo biotinylation. A portion of both Snat3 and Snat1 vascular expressions was localized on luminal membranes. Importantly, Snat1 expression was restricted to larger cortical microvessels, whereas Snat3 was additionally expressed on BBB capillary membranes. This differential expression of system A (Snat1) versus system N (Snat3) transporters suggests distinct roles for Snats in the cerebral vasculature and is consistent with Snat3 involvement in net transendothelial BBB AA transport.     

Regulation of ABC membrane transporters in glial cells: relevance to the pharmacotherapy of brain HIV-1 infection

Limited drug penetration is an obstacle that is often encountered in the treatment of CNS diseases including human immunodeficiency virus type-1 (HIV-1) encephalitis (HIVE). One mechanism that may contribute to this phenomenon is the expression of ATP-binding cassette (ABC) drug efflux transporters [i.e., P-glycoprotein (P-gp), Multidrug Resistance-Associated Proteins (MRP/Mrp), Breast Cancer Resistance Protein (BCRP; also known as ABCG2)] at the primary brain barrier sites (i.e., blood-brain barrier, blood-cerebrospinal fluid barrier). In addition, it has been recently proposed that glial cells may also contribute to the low accumulation and altered distribution of therapeutic compounds in the CNS by functioning as a "secondary barrier." In fact, a few studies have shown that ABC transporters are both expressed and functional in glial cells. Furthermore, commonly prescribed antiretroviral compounds (ARVs), particularly HIV-1 protease inhibitors, are substrates for many of these same transport proteins suggesting that ABC transporters in glial cells may contribute to the overall export of these drugs from the brain. HIV-1 infection is a chronic condition characterized by long-term exposure of brain cellular compartments to HIV-1 virions and soluble viral proteins. In addition, treatment of HIV-1 infection involves long-term administration of a multiplicity of ARVs (i.e., HAART regimens). Indeed, pathological factors associated with HIV-1 infection and/or pharmacological factors related to treatment may alter the expression of ABC transporters and lead to changes in CNS ARV uptake and/or distribution. This review summarizes recent knowledge in this area and emphasizes the role that glial ABC transporters may play in regulating ARV transport.     

Aluminum citrate uptake by immortalized brain endothelial cells: implications for its blood-brain barrier transport

The objective was to further test the hypothesis that aluminum (Al) citrate transport across the blood–brain barrier is mediated by a monocarboxylate transporter (MCT). Speciation calculations showed that Al citrates were the predominant Al species under the conditions employed. Al citrate did not inhibit lactate uptake and was not taken up by the rat erythrocyte, suggesting it does not serve as an effective substrate for either MCT1 or the anion exchanger. Studies were conducted with b.End5 cells derived from mouse brain endothelial cells to identify the properties of the carrier(s) mediating Al citrate transport. Western blot analysis of b.End5 cells showed expression of the transferrin receptor and MCT1, but not MCT2 or MCT4. Uptake of Al citrate was 70% faster than citrate. Citrate and Al citrate uptake were sodium independent. Citrate uptake increased at pH 6.9 compared to 7.4, whereas Al citrate uptake did not. Al citrate uptake was reduced by inhibitors of mitochondrial respiration and oxidative phosphorylation, suggesting ATP dependence, but not by ouabain, suggesting no role for Na/K-ATPase. Uptake was not affected by -ketoglutarate or malonate, substrates for the dicarboxylate carrier. Many substrates and inhibitors of MCT1 and organic anion transporters reduced Al citrate uptake into b.End5 cells. BSP and fluorescein, organic anion transporter substrates/inhibitors, inhibited Al citrate uptake. We conclude that Al citrate transport across the blood–brain barrier is carrier-mediated, involving either an uncharacterized MCT isoform expressed in the brain such as MCT7 or MCT8 and/or one of the many members of the organic anion transporting protein family, some of which are known to be expressed at the blood–brain barrier.     

Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B0AT2 and y+LAT1 functional cooperation

In the CNS, amino acid (AA) neurotransmitters and neurotransmitter precursors are subject to tight homeostatic control mediated by blood-brain barrier (BBB) solute carrier amino acid transporters (AATs). Since the BBB is composed of multiple closely apposed cell types and opportunities for human in vivo studies are limited, we used in vitro and computational approaches to investigate human BBB AAT activity and regulation. Quantitative real-time PCR (qPCR) of the human BBB endothelial cell model hCMEC/D3 (D3) was used to determine expression of selected AAT, tight junction (TJ), and signal transduction (ST) genes under various culture conditions. L-leucine uptake data were interrogated with a computational model developed by our group for calculating AAT activity in complex cell cultures. This approach is potentially applicable to in vitro cell culture drug studies where multiple “receptors” may mediate observed responses. Of 7 Leu AAT genes expressed by D3 only the activity of SLC7A5-SLC3A2/LAT1-4F2HC (LAT1), SLC43A2/LAT4 (LAT4) and sodium-dependent AATs, SLC6A15/B⁰AT2 (B⁰AT2), and SLC7A7/y⁺LAT1 (y⁺LAT1) were calculated to be required for Leu uptake. Therefore, D3 Leu transport may be mediated by a potentially physiologically relevant functional cooperation between the known BBB AAT, LAT1 and obligatory exchange (y⁺LAT1), facilitative diffusion (LAT4), and sodium symporter (B⁰AT2) transporters.