Download Transport of Xenobiotics Across the Blood-Brain Barrier

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.
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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-
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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.
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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.,
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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.
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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)
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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.