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Transport of Xenobiotics Across the Blood-Brain Barrier Bruno Hagenbuch, Bo Gao, and Peter. J. Meier Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland Distinct transport proteins regulate the movement of waste products and xenobiotics across the blood-brain barrier (BBB). Members of the drug transporter families MDR, MRP, and OATP have been identified in the BBB, and a detailed characterization of the involved proteins is now required to target drugs more efficiently to the brain. n the central nervous system there are two fluid barriers: the blood-brain barrier (BBB) formed by the brain capillary endothelial cells and the blood-cerebrospinal fluid barrier formed by the choroid plexus. Capillary endothelial cells that form the BBB are not fenestrated, have minimal pinocytosis, and are connected by high-resistance tight junctions. This reduces the unregulated diffusion of molecules across the BBB to a minimum (15). In addition, the tight junctions separate the plasma membrane into a luminal domain facing the blood side and an abluminal domain facing the brain side (Fig. 1). Thus capillary endothelial cells form a polarized barrier similar to the polarized barriers located in the small intestine, the renal proximal tubule, or the liver. Functionally, the BBB actively regulates the transport of nutrients, waste products, and drugs into and out of the brain by means of distinct transport systems expressed in the luminal and/or abluminal membrane domain. This is similar to the small intestine and the liver, where multispecific transporters such as the organic anion transporting polypeptides (Oatps in rodents, OATPs in humans), the multidrug resistance protein 1 (Mdr1a/b, MDR1), as well as the multidrug resistance-associated proteins (Mrps/MRPs) have been identified and work in concert with detoxification enzymes to protect the organism from potentially harmful compounds. During the characterization of these drug transporters, individual proteins were also identified in the cells of the BBB. Their role in the transport of xenobiotics across the BBB will be summarized in this review. Drug transporters expressed in choroid plexus epithelial cells have been summarized elsewhere (7). The ATP-dependent efflux pumps MDRs. The MDRs belong to the ATP-binding cassette (ABC) superfamily of transporters and were first identified in mammalian tumor cells, where they conferred multidrug resistance (9). In humans, there are two proteins, known as MDR1 and MDR3 (also called MDR2), whereas in rodents there are three forms, Mdr1a, Mdr1b, and Mdr2 (Table 1). The human MDR3 and the rodent Mdr2 are probably not involved in multidrug resistance. They are phospholipid flippases expressed at the canalicular membrane of hepatocytes, where they are important for the secretion of phosphatidylcholine into bile. However, human MDR1 and rodent Mdr1a/b confer multidrug 0886-1714/02 5.00 © 2002 Int. Union Physiol. Sci./Am. Physiol. Soc. www.nips.org resistance by actively exporting a wide variety of mainly amphipathic and hydrophobic compounds from tumor cells. Under normal physiological conditions, these efflux pumps are expressed in organs involved in the elimination of endoand xenobiotics, such as the liver and the kidney, and in epithelial tissues that protect the organs from entry of xenobiotics, like the small intestine, testes, placenta, and BBB (17). Schinkel and coworkers (18) demonstrated the expression of Mdr1a in the capillary endothelial cells of the BBB and its physiological importance in protecting the brain from xenobiotics by using knockout mice. By immunohistochemistry, Mdr1 was identified in capillary endothelial cells of normal but not of Mdr1a-deficient mice and later was fine-localized to the luminal membrane of these cells (3, 8), where it can mediate efflux of potentially toxic compounds. The protective function of Mdr1 was discovered because the Mdr1a-deficient mice demonstrated a much higher sensitivity to ivermectin, a neurotoxin used to treat mite infestations and normally tolerated very well because of its inability to cross the BBB. Compared with the wild-type mice, the Mdr1a-deficient mice were 50- to 100-fold more sensitive to ivermectin, and, on treatment with subtoxic amounts, the brain levels in the knockout mice were ~90-fold higher compared with normal mice. Similarly, the brain level ratio between Mdr1a-deficient and normal mice was elevated for numerous additional compounds, including HIV protease inhibitors (amprenavir, nelfinavir, indinavir, and saquinavir), immune suppressants (tacrolimus and cyclosporin), anticancer drugs (vinblastine and paclitaxel), and the antiarrythmic quinidine as well as the glucocorticoid dexamethasone and the cardiac glycoside digoxin (2). These results demonstrate the importance of Mdr1a for brain protection from drug toxicity but also suggest that coadministration of a specific Mdr inhibitor (also called a reversal agent) could enhance penetration of the BBB by certain compounds. To test this possibility, Mayer and coworkers (12) used a very effective reversal agent, PSC833, a nonimmunosupressive cyclosporin A analog, and determined brain levels of digoxin (12). Oral administration of PSC833 to wild-type mice 2 h before an intravenous injection of [3H]digoxin increased brain digoxin levels 19-fold compared with wild-type mice not receiving PSC833. However, brain digoxin levels in Mdr1a-knockout mice were even higher, suggesting that PSC833 administration resulted in a significant but not com- Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on May 2, 2017 I News Physiol Sci 17: 231 234, 2002; 10.1152/nips.01402.2002 231 FIGURE 1. Schematic representation of the capillary endothelial cells that build up the blood-brain barrier (BBB). A: cross-section through a brain capillary. B: longitudinal section across the wall of the capillary, with the tight junctions that separate the membranes into the luminal and abluminal domains. The OATPs The Oatps/OATPs are a group of membrane transporters classified within the solute carrier family 21A (rodents, Slc21a; human, SLC21A) (Table 2). They exhibit a wide spectrum of TABLE 1. Multispecific ATP-dependent drug transporters Multidrug Resistance Proteins Human Protein Gene Symbol Mouse Protein Gene Symbol Rat Protein Gene Symbol MDR1 ABCB1 Mdr1a Abcb1a Mdr1a Abcb1 Mdr1b Abcb1b Mdr1b Abcb1b Abcb4 Mdr2 Abcb4 MDR3/2 ABCB4 Mdr2 Human Protein Gene Symbol Mouse Protein Gene Symbol Rat Protein Gene Symbol MPR1 ABCC1 Mrp1 Abcc1a Mrp1 Abcc1 MRP2 ABCC2 Mrp2 Abcc2 Mrp2 Abcc2 MRP3 ABCC3 Mrp3 Abcc3 Mrp3 Abcc3 MRP4 ABCC4 Mrp4 Abcc4 Mrp4 Abcc4 MRP5 ABCC5 Mrp5 Abcc5b MRP6 ABCC6 Mrp6 Abcc6 Mrp6 Abcc6 MRP7 ABCC10 Mrp7 Abcc10 Multidrug Resistance-Associated Proteins The transporter proteins shown in bold have been identified at the blood-brain barrier. 232 News Physiol Sci • Vol. 17 • December 2002 • www.nips.org Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on May 2, 2017 plete inhibition of Mdr1a-mediated digoxin export across the BBB. These results confirm that 1) Mdr1a plays an important role in protecting the brain from various compounds by preventing their penetration of the BBB and 2) that specific reversal agents might be promising compounds to overcome this limitation and increase the brain levels of certain drugs. MDR-associated proteins. Besides the MDRs, there are other drug efflux pumps that also belong to the ABC transporters. The first member was originally cloned from a multidrug-resistant human lung cancer cell line and therefore named multidrug resistance-associated protein (MRP). The MRP family contains at least seven members, MRP1 to MRP7 (4), with MRP1 and MRP2 being the best characterized (Table 1). The ubiquitously expressed MRP1 is the major leukotriene C4 (LTC4) transporter. When overexpressed, MRP1 confers resistance to different antitumor agents such as vincristine and daunorubicin. Under normal physiological conditions, however, MRP1 helps to protect the organism against toxic compounds, as was demonstrated by the Mrp1-knockout mice that are hypersensitive to the anticancer drug etoposide (16). MRP2, also known as canalicular multispecific organic anion transporter (cMOAT), is strongly expressed in liver, kidney, and small intestine. Deficiency of MRP2 results in the Dubin-Johnson syndrome, a genetically inherited disease that is characterized by impaired excretion of glucuronidated bilirubin, which results in conjugated hyperbilirubinemia (10). So far, no Mrp2-knockout mice have been generated. However, Mrp2-deficient rat strains, like the transport-negative TR rats and the Eisai hyperbilirubinemic rats, are useful animal models and have been extensively studied to determine the substrate specificity of the canalicular Mrp2. It turned out that Mrp2 is an important component of the detoxification system of hepatocytes and that it excretes anionic glucuronides as well as glutathione conjugates of endo- and xenobiotics, including many drugs and drug conjugates but also unconjugated organic anions, into bile (11). Recently, Miller and coworkers (14) were able to show that Mrp2 is expressed at the BBB in isolated rat brain capillaries by using functional experiments and confocal microscopy. They demonstrated that luminal accumulation of the fluorescent organic anion and Mrp substrate sulforhodamine 101 was inhibited by coincubation of the capillaries with other Mrp substrates such as LTC4 and that MDR1-specific inhibitors like the reversal agent PSC833 had no effect. Using an antiMrp2 antibody, confocal immunolocalization studies detected Mrp2 at the luminal surface of the endothelium of normal rats, but no staining was obtained with capillaries isolated from the mutant TR rats. Thus Mrp2 is clearly expressed at the rat BBB, and, given the expression of the drug-metabolizing enzymes in the endothelial cells, it might play a similar important role in the detoxification and protection of the brain as in hepatocytes. In addition, the effect of the HIV protease inhibitors saquinavir and ritonavir on Mdr1- and Mrp2-mediated transport was tested, and it could be demonstrated that both agents interact with both ABC transporters and therefore might be helpful reversing agents for drug resistance caused by both ABC transporters. TABLE 2. Multispecific organic anion transporting polypeptides (Oatp/OATPs) Human Protein Gene Symbol hPGT SLC21A2 OATP-A SLC21A3 OATP-C Mouse Protein Gene Symbol Rat Protein Gene Symbol Oatp1 Slc21a1 Oatp1 Slc21a1 mPGT Slc21a2 rPGT Slc21a2 OAT-K1 Slc21a4 Oatp2 Slc21a5 Oatp2 Slc21a5 Oatp3 Slc21a7 Oatp3 Slc21a7 Oatp9 Slc21a9 Oatp9 Slc21a9 SLC21A6 SLC21A8 OATP-B SLC21A9 Oatp4 Slc21a10 Oatp4 Slc21a10 OATP-D SLC21A11 Oatp11 Slc21a11 Oatp11 Slc21a11 OATP-E SLC21A12 Oatp12 Slc21a12 Oatp-12 Slc21a12 Oatp5 Slc21a13 Oatp5 Slc21a13 Oatp14 Slc21a14 Oatp14 Slc21a14 OATP-F SLC21A14 Only human OATP-A and rat Oatp2 have been identified so far at the blood-brain barrier on the protein level. PGT, prostaglandin transporter. amphipathic transport substrates. Some members are selectively expressed in the liver, where they are involved in the hepatic elimination of endo- and xenobiotics. Most Oatp/OATPs however, are expressed in multiple tissues, including the BBB, choroid plexus, lung, heart, intestine, kidney, placenta, and testis (20). Many Oatp/OATPs represent polyspecific organic anion transporters with partially overlapping and partially distinct substrate specificities for a wide range of amphipathic organic solutes, including bile salts, organic dyes, steroid conjugates, thyroid hormones, neuroactive peptides, numerous drugs, and other xenobiotics (13). Two of the best-characterized members, human OATP-A (SLC21A3) and rat Oatp2 (Slc21a5), have recently been localized to the BBB by using immunolocalization techniques (6, 8). Human OATP-A was the first human OATP isolated from human liver. Later it turned out that its strongest expression is in brain, followed by kidney, liver, lung, and testis. An OATPA-specific antibody recognized a ~60-kDa protein in human frontal cortex homogenates and stained brain microvessels and capillaries, whereas astrocytes and neurons were immunonegative (6). Similarly, rat Oatp2, which was isolated from rat brain, was exclusively localized to endothelial cells of cerebral capillaries by using in situ hybridization as well as immunolocalization with an Oatp2-specific antibody (8). Unlike Mdr1, which is expressed only in the luminal membrane of the endothelium, Oatp2 was detected in both the luminal and the abluminal membranes. These two Oatp/OATPs, which share 73% amino acid identities, have been extensively characterized functionally in different in vitro systems. It was demonstrated that they transport similar compounds, including sulfated and glucuronidated steroids [dehydroepiandrosterone sulfate (DHEAS), estrone-3sulfate, and estradiol-17--glucuronide], thyroid hormones (T3 and T4), drugs like fexofenadine, cationic compounds (ADPajmalinium, rocuronium), and neuroactive peptides {[D- penicillamine 2,5]enkephalin (DPDPE)}. In addition, there are compounds that are only transported by either OATP-A (e.g., Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on May 2, 2017 OATP8 deltorphin II and the cyanobacterial toxin microcystin) or Oatp2 (e.g., Leu-enkephalin and digoxin) (13). That Oatp2 indeed is functional at the BBB has been demonstrated by different in vivo studies in which transport of the Oatp2 substrates DHEAS, estradiol-17--glucuronide, and DPDPE across the rat and mouse BBB was determined either by injection of radiolabeled compound directly into the cortex or by an in situ brain perfusion technique. Asaba et al. (1) studied transport of the neuroactive steroid DHEAS across the rat BBB and could show that apparent efflux was 10-fold higher than influx and was saturated, with an apparent Km value of 33 2M, which is similar to the 17 2M determined for Oatp2-mediated DHEAS transport in vitro. This net DHEAS efflux, which is physiologically the right transport direction since DHEAS concentrations in rodent brain by far exceed the concentrations in the periphery, could be inhibited by several Oatp2 substrates. Furthermore, by using immortalized mouse brain capillary endothelial cells in culture, uptake of DHEAS (apparent Km ~ 34 2M) and digoxin could be demonstrated. By using RT-PCR FIGURE 2. Multispecific drug transporters expressed in the BBB. Only transporters that have been localized in the BBB at the protein level are indicated, with examples of transport substrates mentioned in the text. News Physiol Sci • Vol. 17 • December 2002 • www.nips.org 233 Summary and perspectives Of the three drug transporter families MDR, MRP, and OATP, so far only four individual members have been localized in the BBB at the protein level (Tables 1 and 2). The only protein shown to be expressed in the human BBB is OATP-A. In the rat, immunostaining of capillary endothelial cells has been obtained with antibodies against Mdr1, Mrp2, and Oatp2. Whereas Mdr1 and Mrp2 are located exclusively in the luminal membrane of the endothelial cells (Fig. 2), Oatp2 is expressed in both the luminal and the abluminal membrane (Fig. 2). Under normal physiological conditions, this arrangement of transporters with a bidirectional Oatp2 in both membranes and the unidirectional Mdr1 and Mrp2 in the luminal membrane results functionally in an efficient efflux system for compounds that need to be secreted from the brain, as exemplified by estradiol-17--glucuronide or DHEAS, as well as an efficient protection system for the brain from uptake of potentially toxic solutes such as, for example, digoxin. What does this mean for transport of drugs across the BBB? Given that many drugs are substrates for the discussed drug transporters, it is evident that efficient efflux systems prevent certain drugs from reaching the brain. To overcome the tight BBB, all involved transport proteins expressed in the human BBB need to be unequivocally identified and characterized in detail. By modeling compounds to be specific substrates, e.g., for Oatp2 but not recognized by Mrp2, and coapplication of reversal agents to inhibit Mdr1, it might be possible in the future to target drugs with higher efficiency to the brain and thus to better treat the numerous disorders of the central nervous system, where currently no or only inefficient drugs are available. This work was supported by the Swiss National Science Foundation (Grants 31-59204.99 to B. Hagenbuch and 31-64140.00 to P. J. Meier) 234 News Physiol Sci • Vol. 17 • December 2002 • www.nips.org References 1. Asaba H, Hosoya K, Takanaga H, Ohtsuki S, Tamura E, Takizawa T, and Terasaki T. Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2. J Neurochem 75: 1907 1916, 2000. 2. Ayrton A and Morgan P. Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 31: 469 497, 2001. 3. Beaulieu E, Demeule M, Ghitescu L, and Beliveau R. P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. Biochem J 326: 539 544, 1997. 4. Borst P, Evers R, Kool M, and Wijnholds J. The multidrug resistance protein family. Biochim Biophys Acta 1461: 347 357, 1999. 5. Dagenais C, Ducharme J, and Pollack GM. Uptake and efflux of the peptidic delta-opioid receptor agonist [D-penicillamine2,5]-enkephalin at the murine blood-brain barrier by in situ perfusion. Neurosci Lett 301: 155 158, 2001. 6. Gao B, Hagenbuch B, Kullak-Ublick GA, Benke D, Aguzzi A, and Meier PJ. Organic anion-transporting polypeptides mediate transport of opioid peptides across blood-brain barrier. J Pharmacol Exp Ther 294: 73 79, 2000. 7. Gao B and Meier PJ. Organic anion transport across the choroid plexus. Microsc Res Tech 52: 60 64, 2001. 8. Gao B, Stieger B, Noè B, Fritschy JM, and Meier PJ. Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain. J Histochem Cytochem 47: 1255 1264, 1999. 9. Juliano RL and Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455: 152 162, 1976. 10. Kartenbeck J, Leuschner U, Mayer R, and Keppler D. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology 23: 1061 1066, 1996. 11. König J, Nies AT, Cui Y, Leier I, and Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1461: 377 394, 1999. 12. Mayer U, Wagenaar E, Dorobek B, Beijnen JH, Borst P, and Schinkel AH. Full blockade of intestinal P-glycoprotein and extensive inhibition of blood-brain barrier P-glycoprotein by oral treatment of mice with PSC833. J Clin Invest 100: 2430 2436, 1997. 13. Meier PJ, Eckhardt U, Schroeder A, Hagenbuch B, and Stieger B. Substrate specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver. Hepatology 26: 1667 1677, 1997. 14. Miller DS, Nobmann SN, Gutmann H, Toeroek M, Drewe J, and Fricker G. Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol 58: 1357 1367, 2000. 15. Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Disc 1: 131 139, 2002. 16. Rappa G, Finch RA, Sartorelli AC, and Lorico A. New insights into the biology and pharmacology of the multidrug resistance protein (MRP) from gene knockout models. Biochem Pharmacol 58: 557 562, 1999. 17. Schinkel AH. The physiological function of drug-transporting P-glycoproteins. Semin Cancer Biol 8: 161 170, 1997. 18. Schinkel AH, Smit JJM, Vantellingen O, Beijnen JH, Wagenaar E, Vandeemter L, Mol CAAM, Vandervalk MA, Robanusmaandag EC, Teriele HPJ, Berns AJM, and Borst P. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77: 491 502, 1994. 19. Sugiyama D, Kusuhara H, Shitara Y, Abe T, Meier PJ, Sekine T, Endou H, Suzuki H, and Sugiyama Y. Characterization of the efflux transport of 17-estradiol-D-17--glucuronide from the brain across the blood-brain barrier. J Pharmacol Exp Ther 298: 316 322, 2001. 20. Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, and Tsuji A. Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Biochem Biophys Res Commun 273: 251 260, 2000. Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on May 2, 2017 and sequencing, an Oatp2-like transcript was identified, suggesting that BBB Oatp2 mediates efflux of DHEAS also in the mouse. A similar study by Sugiyama et al. (19) characterized estradiol-17--glucuronide efflux across the rat BBB. With specific inhibitors for the different organic anion transporters, they deduced that the major part of estradiol-17--glucuronide efflux was mediated by Oatp2. In a third study, Dagenais et al. (5) investigated transport of the /-opioid receptor agonist DPDPE across the BBB of normal and Mdr1a-knockout mice by using a brain perfusion technique. Due to Mdr1a-mediated efflux, DPDPE exhibited poor BBB permeability in wild-type mice, whereas in the Mdr1a-knockout mice uptake of DPDPE could readily be determined. The obtained results suggest that Oatp2 is responsible for saturable uptake of DPDPE and potentially other opioid peptides across the BBB into the brain. Together, these results show that Oatp2 and OATP-A are expressed at the BBB and functionally can mediate either 1) efflux of compounds that are synthesized in the brain and active in the periphery or waste products that need to be excreted or 2) uptake of potentially neuroactive compounds such as, for example, opioid peptides.