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Novel and emerging strategies in drug delivery
for overcoming the blood–brain barrier
Two decades of molecular research have revealed the presence of transporters and receptors expressed in the
brain vascular endothelium that provide potential novel targets for the rational design of blood–brain barrierpenetrating drugs. In this review, we briefly introduce the reader to the molecular characteristics of the blood–brain
barrier that make this one of the most important obstacles towards the development of efficacious CNS drugs.
We highlight recent attempts to rationally target influx and bidirectional transport systems expressed on the brain
endothelial cell and avoid the important obstacle presented in the form of efflux transporters. Many of these
approaches are highly innovative and show promise for future human application. Some of these approaches,
however, have revealed significant limitations and are critiqued in this review. Nonetheless, these combined efforts
have left the field of CNS drug delivery better positioned for developing novel approaches towards the rational
design of CNS‑penetrating drugs.

Blood–brain barrier
The blood–brain barrier (BBB) is a highly
dynamic capillary endothelial interface between
blood and brain. The blood–cerebrospinal fluid
barrier resides within the choroid plexus epithelium. While both can be targeted for drug delivery into the CNS, little is known about drug
transport across the choroid plexus, whereas the
BBB is the major target of such research efforts.
In this review, we will therefore focus on the
brain capillary endothelium as a target organ
for CNS drug delivery.
At the BBB, influx transporters facilitate
uptake of ions, amino acids, glucose and other
nutrients from the blood to meet the high nutrient and energy demand of the brain. The BBB
protects the brain from chemical and bacterial
assault, while simultaneously permitting the passage of nutrients required for metabolic brain
functions. As a result, the BBB limits the systemic
transport of drugs into the brain for the treatment
of neurologic disease [1] . The BBB is comprised of
brain microvascular endothelial cells (BMECs)
fused together by high-resistance tight junctions
[2] . Pericytes, astrocytic foot processes and neuronal processes cover most of the abluminal membrane (brain side) of the BMECs. Together, these
interactive cells form the neurovascular unit.
Paracellular transport of neurotherapeutics into
the brain is blocked by the high-resistance tight
junctions joining the BMECs. Drugs can either
pass through the BMECs via simple diffusion or
facilitated transport. The restrictive permeability

of the BBB is tightly controlled by the BMECs
through the regulated expression of carrier-mediated and receptor-mediated transport systems in
the BMEC luminal (blood side) and abluminal
membranes, combined with the expression and
activity of multiple efflux transport systems.
Carrier-mediated transport of solutes is facilitated by specific substrate–transporter interactions and driven by direct energy conversion
(e.g., ATP hydrolysis), concentration gradients,
cotransport or various combinations of the above.
BBB transporters have been identified for amino
acids, sugars, nucleosides, cytokines, monocarboxylic acids, peptides, organic cations and
organic anions [3] and include glucose transporters (GLUTs), monocarboxylic acid transporters
[4] , peptide transporters [5] , organic anion transporters (OATs), organic cation transporters
and organic anion transporting polypeptides
(Oatps). In addition, multiple members of
the ATP-binding cassette (ABC) superfamily,
including P-glycoprotein (P-gp), breast cancer
resistance protein (BCRP1) and multidrug resistance-associated proteins (MRPs), are expressed
at the BBB [6] .

10.4155/FMC.09.137 © 2009 Future Science Ltd

Future Med. Chem. (2009) 1(9), 1623–1641

David D Stenehjem1,
Anika MS Hartz2 , Björn
Bauer1 & Grant W

Author for correspondence
UMD College of Pharmacy, 232
Life Science, 1110 Kirby Drive,
Duluth, MN 55812, USA
Tel.: +1 218 726 6007
UMD Biochem/Molecular
Biology, 252 SMed D602,
1035 University Drive, Duluth,
MN 55812, USA
Blood – brain barrier
Highly dynamic and selective
capillary endothelial interface
separating the circulating blood
from the brain parenchyma
that controls what goes in and
comes out of the CNS. The
endothelium is comprised of
endothelial cells physically
joined together by
tight junctions


delivery across the BBB:
an ongoing challenge
Simple diffusion of a neurotherapeutic is enhanced
by both a high degree of lipophilicity and a molecular weight less than 400–500 Da [7] . Developing
in silico predictors of the chemical characteristics
required for diffusion across the BBB has been the
ISSN 1756-8919


Review | Stenehjem, Hartz, Bauer & Anderson
Influx transporters
Transporter proteins that
function to traffic nutrients into
the brain. Influx transporters
are targeted to deliver
therapeutic drugs into the brain.
Most influx transporters are
bidirectional and can move
substrates in either direction
across a cell membrane, as
determined by molecular
driving forces
Brain microvascular
endothelial cell

Cell lining the lumen of the
brain microvasculature; the
basic unit that forms the
blood–brain barrier

Carrier- mediated transport
Transport of solutes across the
blood–brain barrier facilitated
by transporter proteins
localized in the plasma
membrane. True facilitated
transport is passive (energy
independent), with solute
moving down its
concentration gradient

Receptor- mediated transport
Transport of substrates via
receptor mediated interactions
and delivery of large molecules
across the BBB
through transcytosis

Efflux transporters
Energy-dependent transporter
proteins that function to traffic
xenobiotics in a unidirectional
fashion, usually from the brain
to the blood. These xenobiotics
include a large number of drugs


main strategy used by pharmaceutical companies
to predict the ability of novel compounds to penetrate into the CNS [8] . These predictors indicate
that size and solubility restrictions of diffusion
prevent brain uptake of many drugs including
large and hydrophilic molecules, proteins and antibodies. Additionally, efflux transporters remove
many lipid-soluble, small drugs from the endothelial cell, preventing distribution into the brain [9] .
Strategies have been developed to bypass the BBB
via invasive techniques, such as direct injection
into the brain, infusion or global disruption of the
BBB. However, direct injection or infusion present major procedural safety concerns and are not
applicable to systemic brain disease [10] . Disruption
of the BBB by either hypertonic mannitol [11] ,
alkylglycerol [12] or a bradykinin analogue  [13]
increases BBB permeability (see later), but may
cause permanent brain damage from unintended
molecules entering the brain. Obviously, these
approaches cannot be used globally in designing
novel neuro­therapeutics. The use of less-invasive
nano­particles and liposomes as drug-delivery vehicles to the CNS have also received recent attention
and are reviewed elsewhere [14–17] .
A major limitation in the in silico modeling
favored by pharmaceutical companies is a paucity
of in vitro and in vivo transport data upon which to
build the models, including data regarding both
passive diffusion and facilitated transport. With
the advent of genomic and proteomic research, a
number of facilitated transport systems expressed
in the BMECs have been identified, including
the organic anion and cation, amino acid, mono­
carboxylic acid, thyroid hormone, glucose, nucleoside and peptide transporters introduced earlier.
Knowledge on the localization of these transporters in either the luminal or abluminal membrane
of the endothelial cell provides opportunities to
develop novel drugs that directly target these
transport systems. Importantly, however, the
efflux transport systems must also be taken into
consideration when rationally designing drugs
targeting facilitated transporters as substrates of
these transporters are often also targets of efflux
transport systems. Drug-delivery strategies targeting these transport systems constitute much
of the focus of this review.
Brain drug delivery &
BBB‑targeting strategies
„„Carrier-mediated transport
Carrier-mediated transport (CMT) provides a
facilitated mechanism for certain small molecules, nutrients and hormones to passively cross
Future Med. Chem. (2009) 1(9)

the BBB following a concentration gradient
(Figure 1) . CMT proteins include the organic
anion-transporting polypeptides, the large neutral amino acid, monocarboxylic acid, glucose
organic anion and cation transporters. CMT
transporters are substrate-selective and carry
their substrates via uni- or bi-directional, mono-,
co- or counter-transport. The rate of substrate
transport depends on substrate affinity for the
transporter, molecular driving force, intracellular
binding capacity and, in some instances, efflux
transport activity as these substrates are often
also targets of efflux transport systems [10,18,19] .
To exploit carrier-mediated transport systems
that could facilitate drug delivery into the CNS,
researchers have focused on structural similarities between therapeutic drug molecules and
endogenous transporter substrates [3] . Molecular
cloning of transporters allows the determination of quantitative structure–activity relationships (QSARs) for individual transport systems.
This knowledge, in turn, facilitates the rational
design of novel small-molecule therapeutics
against a target within the CNS and a specific
carrier-mediated transporter. Trojan horse drugs
can also be designed to target specific CMT systems [20] . For these drugs, a neurotherapeutic
is conjugated to an (usually) endogenous CMT
substrate with the CMT substrate driving the
delivery of the neurotherapeutic across the
BBB via the targeted CMT system (Figure 1) .
Examples of neurotherapeutics targeting CMT
systems are now discussed.

neutral amino acid transporter
The large neutral amino acid transporter (LAT1)
is a nutrient transporter that is located in the
luminal and abluminal membranes of the brain
capillary endothelium [21] . At the BBB, LAT1
mediates brain uptake of neutral l-amino acids
such as phenylalanine, leucine and tyrosine
(Figure  2) [22] . Therefore, LAT1 has received
attention for delivering drugs into the brain that
are either similar in structure to LAT1 substrates
or are conjugated to LAT1 substrates. The most
successful example of the former approach is
l-DOPA (3,4-dihydroxy-l-phenylalanine,
Levodopa®), an amino acid derivative that is used
to increase brain dopamine levels in Parkinson’s
disease. While dopamine itself does not cross the
BBB, LAT1 facilitates l-DOPA transport into
the brain, where it is de­carboxylated to active
dopamine. Other CNS drugs transported by
LAT1 include a-methyldopa, baclofen and
gabapentin [23–25] .
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Emerging strategies in drug delivery for overcoming the blood–brain barrier

Trojan horse


| Review

Trojan horse

Endogenous substrates
Novel drugs

Brain capillary
endothelial cell

Tight junction


Neuronal and glial target cells

Future Med. Chem. © Future Science Group (2009)

Figure 1. CMT and RMT of novel drugs targeting the CNS. Trojan horse and other novel
substrates can target both CMT and RMT transport systems. CMT is stereoselective and the
transport rate is dependent on the degree of occupation of the carrier. Transporters may be present
on both the luminal and abluminal membranes and substrates traffic through the intracellular
compartments via a variety of mechanisms. RMT initiates with substrate binding to an extracellular,
luminal receptor. Upon binding, the receptor and substrate are endocytosed, forming an
intracellular transport vesicle. The vesicle is transported to the abluminal membrane and, upon
exocytosis, the substrate is released from the endothelial cell.
CMT: Carrier-mediated transport; RMT: Receptor-mediated transport.

One example of targeting LAT1 with a
Trojan horse drug is described in a study
by Killian et  al. [26] . The authors linked the
LAT1 substrate l-cysteine to 6-mercapto­
purine or 2-methyl-1-propanethiol. l-cysteine
is a relativly low-affinity substrate for LAT1
(K m = 480 µM). However, the affinity of l-cysteine for LAT1 can be increased by increasing the substrate’s side-chain lipophilicity [22] .
The anticancer drug 6-mercaptopurine is a
heterocyclic molecule that is relatively polar
compared with the highly lipophilic alkane
2-methyl-1-propanethiol, which is structurally
similar to the side chain of the high-affinity
LAT1 substrate l-leucine. Using in situ brain
perfusion with [14C]-l-leucine as a competitive
LAT1 substrate, Killian et al. showed that both
the 6-mercaptopurine and the 2-methyl-1-propanethiol conjugate targeted LAT1 (K i from
parietal cortex for 2-methyl-1-propanethiol–
l-cysteine = 11.8 µM) [22] . However, due to
higher lipophilicity, the 2-methyl-1-propanethiol conjugate exhibited an increased LAT1
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affinity compared with the 6-mercaptopurine
conjugate, which has been reported previously
with other substrates [22] .
Gynther et al. used a similar Trojan horse
approach to target LAT1 for drug delivery [27] .
Specifically, the authors coupled the hydrophilic, NSAID ketoprofen to the phenolic
hydroxyl group of l-tyrosine [27] . The coupling
reaction resulted in a zwitterionic prodrug where
tyrosine and ketoprofen are linked through an
ester bond. The rationale for creating the ester
linkage is to facilitate release of the conjugated
drug through the actions of esterases present in
the brain parenchyma, although the authors did
not assess enzymatic release of ketoprofen in the
periphery or CNS. Importantly, the conjugation left the l-tyrosine carboxyl and a-amino
groups unsubstituted, allowing recognition by
LAT1 and delivery of the conjugate into the
brain [28] . Using in situ brain perfusion, they
demonstrated that the conjugate was delivered
into the brain and that transport was saturable
and concentration dependent (K m = 22 µM).


Review | Stenehjem, Hartz, Bauer & Anderson
These new data are of considerable interest as
LAT1 continues to prove a useful target for
CNS-penetrant drugs.
Interestingly, Kobayashi et al. recently found
that LAT1 protein and mRNA expression levels
are increased in high-grade gliomas, which is
a likely response to increased nutrient demand
during tumor proliferation [29] . Such a finding is
intriguing as it suggests that neuro­therapeutics
targeting LAT1 may be used to target highgrade gliomas. As an additional benefit, hijacking LAT1-facilitated transport for delivering
chemotherapeutics may also reduce the tumor’s
nutrient supply.

transporter 1
Glucose transporter 1 is the dominant
BMEC glucose transporter and is expressed
at high levels in both the luminal and abluminal membranes [30] . It is noteworthy that
GLUT1 is highly specific for d-glucose transport and only minimal structural changes are
allowed for GLUT1 substrate recognition [31] .
Consequently, targeting GLUT1 with chemotherapeutics conjugated to glucose has been
largely unsuccessful. In two published studies, glucose was coupled to either chlorambucil
[32] or busulfan [33] , but these conjugates were
not transported and even inhibited GLUT1dependent glucose transport. In another study,
a conjugate of l-serinyl-b-d-glucoside with
[Met5]enkephalin produced analgesia in mice,
whereas the unglycosylated enkephalin peptide
did not [34] . While glycosylation reduced the
peptide’s lipophilicity, it also increased BBB
permeability, suggesting GLUT1-facilitated
transport. Similar results were shown with
glycosylated deltorphin and dermorphin analogues [35] and Leu-enkephalin [36] . However,
these data do not directly demonstrate GLUT1dependent transport of the tested therapeutic.
Indeed, more recent work suggests glycosylated
opioids may directly penetrate the BBB via an
endocytotic mechanism [37] . This hypothesis is
derived from data suggesting that glycosylated
opioids directly interact with the lipid bilayer
and, therefore, may transcytose through the
endothelial cell [38] .

anion transporting polypeptides
Organic anion transporting polypeptides provide intriguing targets for the rational design of
CNS drugs as they transport a wide variety of
endo- and xeno-biotic substrates (Figure 2) [39] .
Such promiscuity in substrate recognition is

Future Med. Chem. (2009) 1(9)

useful in drug design, as compounds exhibiting significant structural diversity can be targeted to this family of transporters. Endobiotics
transported by Oatps include bile acids, conjugated sterols, the opioid peptides enkephalin
and deltorphins and one of the most prescribed
drugs on the market today, thyroxine. Other
drugs transported by Oatps include compounds as structurally diverse as methotrexate, saquinavir, atorvastatin, glyburide and the
fenamate class of NSAIDs [39,40] . Hagenbuch
and Gui have recently published a review on
this topic [41] . Structure–activity studies have
been carried out for certain Oatps using limited data sets of specific substrates. In general,
Oatp substrates have a relatively high molecular
weight and usually contain a negatively charged
group, an extended hydrophobic domain in
the central part of the structure and a hydrogen bond donor at the opposite end [42–45] .
Interestingly, some Oatps, including the brain
barrier-expressed Oatp1c1, appear to possess
multiple substrate-binding sites [46] , thus providing additional substrate-binding sites for
targets with rationally designed drugs.
Multiple Oatps are expressed on brain
endothelial cells in both rodents and humans.
In humans, OATP paralogs OATP1C1,
OATP1A2 and OATP2B1 are expressed in
brain endothelial cells [47–49] . In rodents, orthologs (Oatp1c1, Oatp1a4 and Oatp2b1) for each
of these human transporters are also expressed
in brain endothelial cells [49–51] (by convention, Oatp refers to both rodent transporters
and organic anion transporting polypeptides
in general, while OATP refers to human transporters). As the role of solute carriers is to
transport solutes across biological membranes,
determining whether these transporters are
localized to the brain endothelial cell luminal
or abluminal membrane is of key importance.
Of the Oatps expressed in the human and
rodent brain endothelial cell, immunohistochemical (IHC) data suggests localization of
rodent Oatp1c1 and Oatp1a4 at both luminal
and abluminal membranes [49,50,52] . Human
OATP1C1 membrane localization is less clear
as the transporter is expressed on both membranes in some human brain samples but only
abluminally in others [49] . IHC data further
suggests OATP2B1 and OATP1A2 localization
to luminal membranes [47] . Thus, Oatps likely
are involved in bidirectional solute transport
across both luminal and abluminal membranes
of the brain endothelial cell.
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Emerging strategies in drug delivery for overcoming the blood–brain barrier






| Review











NH 2








NH 2






















NH 2






H 2N



NH 2





Figure 2. Select carrier-mediated transport and efflux transporter substrate structures. Organic anion transporting
polypeptide substrates (thyroxine, glyburide, saquinavir, methotrexate and pravastatin), l-amino acid transporter-1 substrates (thyroxine,
tyrosine, leucine and l-DOPA), glucose transporter-1 substrate (glucose), P-glycoprotein substrates (thyroxine, saquinavir and
pravastatin), multidrug-resistance protein-2 substrate (methotrexate). These structures were chosen to illustrate the diversity of
structures recognized by CMT systems and to provide examples of well-described substrates for each transporter. Importantly, many
substrates of CMT systems are also substrates for efflux transporters.

Receptor-mediated transport
Brain microvascular endothelial cells also
express receptor-mediated transport (RMT)
systems that facilitate delivery of large molecules into the brain through transcytosis. This
process is initiated through substrate binding
to a cognate receptor expressed on the luminal
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BMEC membrane (Figure 1) . The bound substrate then undergoes receptor-mediated endocytosis forming intracellular transport vesicles.
Vesicles either dispose of their contents via
lysosomal or ubiquitin–proteasome pathways
or are transported intracellularly to the abluminal side of the BMECs for exocytosis into


Review | Stenehjem, Hartz, Bauer & Anderson
the brain parenchyma. Receptor-mediated
systems can theoretically transport substrates
of large molecular weight, including proteins,
antibodies, genes, enzymes, liposomes, nanoparticles and drugs, in comparison with the more
size-restricted stereos­elective, carrier-mediated
transport systems. RMT systems most widely
explored for CNS drug delivery include the diphtheria toxin receptor and the transferrin, insulin
and low-density lipoprotein (LDL) receptors.

The transferrin receptor is a transmembrane
glycoprotein with two subunits, each of which
binds one transferrin molecule [53] . This receptor is expressed on the luminal surface of
BMECs [54,55] . The receptor delivers iron to the
brain through interactions with transferrin, a
circulating iron-binding protein [56] . Transferrin
is a poor candidate for carrying conjugated
therapeutics since, under normal physiologic
conditions, transferrin receptors are mostly saturated with endogenous transferrin [57] . Research
efforts have therefore focused on generating antibodies raised against the transferrin receptor to
carry conjugated drugs into the brain.
Currently, no humanized or chimeric transferrin antibodies have been developed for CNS
drug targeting in humans or primates. OX26 is
a mouse monoclonal antibody raised against an
extracellular epitope of the rat transferrin receptor [55,58] and is used as a drug-delivery model
in the rat. R17217 and 8D3 are rat monoclonal
antibodies directed against the mouse transferrin
receptor and are used as drug-delivery models in
the mouse [59,60] . A chimeric rat 8D3 monoclonal
has also been produced [61] , possibly allowing
delivery of therapeutic fusion proteins to the
mouse brain. A variety of therapeutics, including
small molecules, proteins and nucleic acids, have
all been delivered into the brain parenchyma by
transferrin receptor-mediated transport. A brief
review of the most recent advances is provided
below and a comprehensive review is provided
elsewhere [57,62,63] .
The OX26 antibody is the most extensively studied transferrin receptor-mediated
drug delivery vector. Recently, OX26 was
coupled to brain-derived neurotrophic factor
(BDNF) for the treatment of ischemic stroke
in a rat model [64] . Avidin–biotin chemistry was
employed to link purified, biotinylated BDNF
protein to OX26 [65] . Streptavidin was coupled
to OX26 by a thiol-ether linkage. The resulting
conjugate improved motor function by 243%

Future Med. Chem. (2009) 1(9)

and significantly reduced stroke volume in rats
with middle cerebral artery occlusion [64] . Other
neurotherapeutic proteins coupled to OX26 have
shown pharmacologic activity in the rat brain,
including vasoactive intestinal protein [66] ,
human basic fibroblast growth factor [67] and
nerve growth factor [68] .
OX26 has also been used to deliver PEGylated
liposomes containing plasmid DNA encoding
either b-galactosidase [69] , tyrosine hydroxylase [70] or short hairpin RNA [71] to the rat brain.
Recently, OX26 PEGylated liposomes carrying
a glial-derived neurotrophic factor expression
vector under the transcriptional control of the
rat tyrosine hydroxylase promoter were tested in
rats with experimental Parkinson’s disease [72] .
A sustained, but modest, therapeutic effect was
observed. OX26 has also been used to deliver
small molecules into the CNS. Loperamide [73]
and daunomycin [74] , two therapeutic drugs that
normally do not, or only at very low levels, cross
the capillary endothelium, have been delivered
across the BBB by OX26 conjugated to liposomes
carrying these therapeutic agents. However,
none of these antibodies recognize the human
transferrin receptor and, therefore, these therapeutic agents are limited to proof-of-principle
studies in animal model systems.

The insulin receptor is expressed on the luminal
membrane of BMECs and also undergoes RMT
[75] . Upon insulin binding to the receptor, a conformational change occurs, promoting endosomal
receptor internalization. Insulin is not considered
as a viable RMT vector due to a short serum
half-life and a potential for inducing hypoglycemia  [76] . A murine monoclonal antibody generated against the human insulin receptor (83-14)
has been tested as a RMT vector in a Rhesus
monkey model system [77] . Both a chimeric [78]
and a fully humanized [79] monoclonal antibody
have also been engineered. Chimeric fusion proteins have been generated that carry glial-derived
neurotrophic factor [80] , BDNF [81] , the lysosomal
enzyme iduronidase [82] , the organophosphatase
paraoxonase [83] and a single-chain Fv antibody
directed against amyloid-b [84] . In all these
cases, the fusion proteins retained their ability
to bind the insulin receptor and interacted with
the secondary target. BDNF, iduronidase and
single-chain Fv fusion proteins are all transported across the Rhesus monkey BBB in vivo.
In a recent study, intravenous administration
of the iduronidase fusion protein to an adult
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Emerging strategies in drug delivery for overcoming the blood–brain barrier
Rhesus monkey resulted in 1% of the injected
dose delivered to 100 g of primate brain [82] . The
authors speculated that the amount of iduronidase delivered to the brain of the Rhesus monkey
could provide sufficient enzyme activity to treat
individuals affected by the lysosomal enzyme
disorder Hurler’s syndrome.

receptor family
The LDL receptor (LDLR) and LDLR-related
proteins 1 and 2 (LRP1 and 2) of the lipoprotein receptor family are also well-studied targets for RMT-mediated drug delivery into the
CNS. LDLR regulates cholesterol homeostasis
by binding apolipoprotein particles, including
ApoE- and ApoB-containing particles, on the
cell surface in the periphery and trafficking
the complex to the lysosome. At the BBB, the
LDLR likely plays a different role and shuttles
apolipoprotein particles to the abluminal BMEC
membrane. The BMEC then releases the cholesterol-carrying particles into the parenchyma
for utilization by neurons or astrocytes [76,85,86] .
The LRP1 and 2 (megalin or glycoprotein 330)
receptors share structural and functional similarities to the LDLR. LRP1 is expressed in the
brain and the BBB, whereas LRP2 is expressed
in the brain but not the BMEC [87] . LRP1 is also
expressed in glioblastomas and brain metastases of lung, skin and breast cancers, suggesting
that LRP1-mediated drug-delivery strategies
may also target primary and secondary brain
tumors. LRP1, acting as an endocytic receptor, transports lactoferrin and melanotransferrin across the BMEC [88,89] . Melanotransferrin
is an endogenous iron-binding protein that
preferentially distributes into brain tissue after
intravenous injection [89] . LRP1 transports melanotransferrin across the BBB 10–15-times faster
than transferrin or lactoferrin [89] . Importantly,
melanotransferrin exists at low physiological concentrations, except in Alzheimer’s disease where
the protein is elevated [90] . Therefore, endogenous
melanotransferrin should not excessively compete
with exogenously administered melanotransferrin
conjugates from binding to LRP1.
Doxorubicin, an anthracycline glycoside,
is a potent chemotherapeutic agent against
glioma cells in  vitro [91] . However, the efflux
pump P-gp excludes doxorubicin from the
CNS, reducing the in vivo efficacy of the compound. Thus, doxorubicin is an excellent candidate for novel CNS drug-delivery strategies
[92,93] . Human melanotransferrin has recently
been covalently conjugated to doxorubicin for
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| Review

treating intracerebral human brain tumors in
nude mice [94] . Conjugation was achieved by
cross-linking melanotransferrin–N-succinimidyl S-acetylthioacetate and doxorubicin–succinimidyl 4-[N-maleimidomethyl] cyclohexane-1carboxylate derivatives. Doxorubicin conjugated
to melanotransferrin is equally tumoricidal
compared with free doxorubicin in subcutaneous gliomas outside the mouse brain  [94] ,
suggesting that conjugation does not impact
the chemotherapeutic activity of doxorubicin.
Importantly, conjugated doxorubicin prolongs
the survival of mice with intracranial gliomas or
mammary tumors compared with unconjugated
doxorubicin, which results in survival similar to
placebo [94] .

One of the most interesting recent approaches
in targeting RMT for CNS drug delivery is the
development of a rationally designed library
of peptides, termed angiopeps, constructed to
shuttle drugs into the brain by targeting LRP
receptors [95] . They are comprised of amino acid
consensus sequences of known human substrates
for LRP1 (e.g., aprotinin, bikunin, amyloid precursor protein and Kunitz-inhibitor precursor),
which possess a Kunitz protease-inhibitor (KPI)
domain. The KPI domain is a structural feature
considered important for recognition, internalization and clearance of amyloid precursor protein by LRP1 [96] . Angiopep-2 is a 19 amino acid
peptide selected from a library of peptides, based
on transport rate assessed by in situ rat brain perfusion and an in vitro BBB model. The apical-tobasolateral transport rate of angiopep-2 is threefold higher than aprotinin, a known substrate for
LRP2, and at least 50-fold higher than transferrin and lactoferrin, other substrates transported across the BBB by RMT. Angiopep-2 is
efficiently transported across the BMEC and is
not recognized by P-gp, good attributes for delivery across the BBB [95] . It is important to note,
however, that it has not yet been directly demonstrated that LRP1 is responsible for angiopep-2
transport across the BBB.
Paclitaxel is an extremely potent chemo­
therapeutic agent against gliomas and brain
metastases in vitro. However, P-gp rapidly eliminates paclitaxel from the brain, limiting treatment of brain tumors in vivo [97–101] . Régina and
colleagues attached three molecules of paclitaxel
to one angiopep-2 molecule by cleavable ester
linkages [102] . Total brain uptake of the paclitaxel–angiopep-2 conjugate was 4.5-fold higher


Review | Stenehjem, Hartz, Bauer & Anderson
than free paclitaxel, as measured by in situ mouse
brain perfusion. The total brain uptake of paclitaxel is an even greater 12–15-fold increase, since
each conjugate contains three paclitaxel molecules. The conjugate retains chemotherapeutic
activity and inherits the ability of angiopep-2
to evade P-gp. Importantly, the conjugate is
active against implanted intracerebral glioblastoma and lung carcinoma tumors in nude mice.
Treatment with paclitaxel–angiopep-2 modestly
prolongs survival compared with vehicle-treated
controls. Currently, paclitaxel–angiopep-2
has entered concurrent Phase I and  II clinical trials in patients with primary or secondary metastatic brain tumors, under the name
ANG1005 (Angiochem).

A nonreplicating lentivirus vector system has
been used to produce LDLR-targeted ApoB
fusion proteins in  vivo [103] . Secreted forms
of glucocerebrosidase or green fluorescence
protein (GFP) were cloned upstream of the
38 amino acid ApoB LDLR-binding domain.
Glucocerebrosidase is the deficient enzyme in the
lysosomal disorder Gaucher’s disease. The nonreplicating lentivirus vector system was administered to mice by intraperitoneal injection. The
recombinant fusion proteins were produced in
the liver and spleen of mice after 14 days and
subsequently secreted into the bloodstream [103] .
Theoretically, once in the bloodstream, the
secreted fusion proteins will bind to the LDLR
on the BMEC surface and transcytose through
the cell for delivery to the brain parenchyma.
In the present study, recombinant protein was
localized to neuronal and astrocyte cell bodies.

an immunological adjuvant for bacterial vaccines
[110,111] . There has been considerable interest in
developing CRM197 Trojan horse compounds
for the delivery of neuro­therapeutics across the
BBB. The biotechnology company To-BBB
Technologies is developing a DTR-mediated
CNS drug-delivery system based on CRM197,
coined 2B-Trans [19,112] . Proof-of-principle studies using intravenous administration of CRM197
conjugated to horseradish peroxidase in a guinea
pig model resulted in the DTR-mediated delivery of the horseradish peroxidase protein across
the BBB [19] .

transport systems
The molecular basis of selective, active barrier
function is a number of multispecific, xenobiotic and drug efflux transporters including
P-gp (ABCB1), breast cancer resistance protein
(BCRP; ABCG2) and multidrug resistance proteins 1, 2, 4 and 5 (MRPs; ABCC family). Of
these, P-gp is the most prominent efflux transporter and research efforts over the last years
have primarily been directed towards this particular transporter. However, in recent years,
BCRP has gained increasing importance at the
BBB. In general, BBB efflux transporters act as
gatekeepers and protect the brain from toxic
compounds but, at the same time, also restrict
drugs from entering the brain (Figure 3) . Thus,
approaches to improve drug delivery to the
brain are circumvention or manipulation of
these transporters. Three strategies have been
developed (Figure 3) :
Rational design of compunds that are not
efflux transporter substrates;


Direct inhibition of efflux transporter function;



toxin receptor
The diphtheria toxin receptor (DTR) is also
known as the membrane-bound precursor of
the heparin-binding EGF-like growth factor
(HB-EGF) [104] . HB-EGF is expressed constitutively on BMECs, neurons and glial cells [105] .
The receptor is upregulated in cerebral blood
vessels under hypoxic conditions (ischemia
or gliomas), inf lammatory conditions and
following seizures [10,106–109] .
The nontoxic mutated diptheria toxin
CRM197 is a ligand for DTR. Upon binding to
the DTR, CRM197 is internalized by receptormediated endocytosis, translocates through the
BMEC cytosol and avoids lysosomal degradation
by utilizing an intrinsic endosomal escape mechanism [10,19,104,109] . CRM197 has been utilized as

Future Med. Chem. (2009) 1(9)

Modulation of efflux transporter expression
and function.



design of compounds that are
not efflux transporter substrates
Given the dominance of P-gp at the BBB,
research has primarily focused on designing drugs to circumvent efflux mediated by
this transporter. Seelig et al. [113] and GatlikLandwojtowicz et al. [114] described a series of recognition elements required for drug–P-gp interaction from analyses of over 100 known P-gp
substrates. The authors found that the number
of clusters of spatially distinct H-bond acceptors
correlated with the strength of the interaction.
Work by Turunen et al. showed that introducing
future science group

Emerging strategies in drug delivery for overcoming the blood–brain barrier


Rational design


Direct inhibition




| Review

Transporter modulation







Future Med. Chem. © Future Science Group (2009)

Figure 3. Modulation of efflux transporters to improve brain drug delivery. (A) Increasing a
drug’s passive diffusion through altering its structure to bypass efflux transporters. (B) Direct
inhibition of drug efflux transporters to increase brain drug delivery. (C) Blocking efflux transporter
intracellular regulation to modulate the transporter’s protein expression and/or transport function
for improved brain uptake of drugs.

anionic and amide structures into paclitaxel, a
P-gp substrate, improved brain uptake, suggesting that this strategy could be used to improve
brain drug delivery of other, structurally complex and lipophilic drugs that are efflux transporter substrates [115] . Some of this information
was later used for rational drug design to avoid
recognition by efflux transporters. Altering the
structure of a compound to increase passive diffusion generally included a 30% reduction on
molecular size, reducing H-bonding groups and
cell partitioning [116] . Overall, however, these
approaches were unsuccessful.

inhibition of efflux
transporter function
Verapamil was the first compound that was
found to block P-gp [117] . Other first generation
P-gp blockers include quinidine, quinine, cyclosporin A, amiodarone and nifedipine [117–120] .
However, these compounds often cause toxic
side effects in vivo due to their low potency, weak
effectiveness and poor selectivity. This led to the
development of new P-gp inhibitors of the second generation, including valspodar (PSC833),
biricodar (VX-710), and third-generation inhibitors, including elacridar (GF120918), tariquidar
(XR-9576), zosuquidar (LY-335979) and laniquidar (R-101933) [119,121–123] . These inhibitors
were primarily developed to overcome drug
resistance in cancer, but only a few inhibitors
have been tested in animals to improve brain
delivery of drugs that are P-gp substrates. For
future science group

example, valspodar was shown to be effective
in increasing brain-drug levels in animal models [122,124,125] . In this regard, Fellner et al. identified P-gp as the major factor in limiting the
anticancer therapeutic paclitaxel from entering
the CNS [98] . They showed that pretreatment
of mice with valspodar increased brain levels
of paclitaxel and, more importantly, reduced
the size of paclitaxel-sensitive, intracerebrally
implanted human glioblastomas by 90%,
whereas paclitaxel by itself did not have an effect.
In a similar study, co-administration of paclitaxel with elacridar and tariquidar in nude mice
led to a long-lasting fivefold increase in paclitaxel
brain levels  [126] . Tariquidar was also used in a
rat PET study to demonstrate inhibition of BBB
P-gp activity in vivo [127] . However, in a study
where tariquidar and the opioid loperamide, a
P-gp substrate, were co-administered in humans,
loperamide did not exert brain opioid effects,
suggesting that tariquidar did not inhibit BBB
P-gp, despite full inhibition of lymphocyte P-gp
[128] . These studies, and others on direct inhibition of BBB P-gp, suggest that transporter
inhibitors often cause toxic side effects in vivo
due to their potency, weak effectiveness and
poor selectivity.
Breast cancer-resistance protein is another
eff lux transporter and was independently
detected at the BBB by several groups [129–131] .
This transporter has recently gained importance, especially in the brain cancer field.
In brain cancer, drug efflux transporters, in


Review | Stenehjem, Hartz, Bauer & Anderson
particular BCRP, underlie multidrug resistance
at the level of the BBB and brain-tumor barrier. An emerging concept also suggests that one
of the major protective mechanisms of cancer
stem cells is anticancer drug efflux mediated by
BCRP and other transporters [132] . This is of
high clinical relevance because, after surgical
removal of the primary tumor, chemotherapy is
the treatment of choice to eliminate cancer cell
and stem cell remnants. However, brain cancer
stem cells overexpressing BCRP do not respond
to chemo­t herapy and can quickly regenerate
into larger and more aggressive tumors. In
addition, recent work showed that drug efflux
transporters, including BCRP and P-gp, at the
BBB and possibly also brain–tumor barrier may
act in concert [133,134] . Based on this novel concept, inhibitors were designed that block two
or more efflux transporters. These so-called
dual inhibitors have primarily been developed
to combat drug resistance mediated by transporters in peripheral tumors and include the
compounds CBT-1 (P-gp and Mrp1 inhibitor
[135] ), as well as nilotinib and sunitinib (both
inhibit P-gp and BCRP [136,137]). It remains to be
seen if these compounds will be effective at the
BBB; however, one example, JAI-51, an inhibitor for BBB P-gp/BCRP, has been shown to
delay glioblastoma growth in mice [138] . At this
point, however, it is unclear whether dual inhibition of P-gp and BCRP is better than selectively
blocking either P-gp or BCRP [99] .
Another recent strategy is to design inhibitors
that show a dual mode of action. One example of
this new type of inhibitor is NSC73306, which
inhibits BCRP and, at the same time, kills P-gpoverexpressing cancer cells [139] . NSC73306 is
currently being tested in BBB models.
Finally, a novel method for inhibiting peptide
efflux by antisense methods has recently been
reported for peptide transport system (PTS)-6
[140] . While efflux pumps for peptides have been
recognized for some time [5] , molecular identification of the responsible transporters has proven
difficult. In this regard, the recent findings of
Dogrukol-Ak and colleagues are of significant
interest [140] . They determined that b-F1 ATPase
acts as the efflux component of PTS-6 and that
antisense targeting b-F1 ATPase increased brain
retention of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) 27
in CD-1 mice. Treatment of a mouse model of
Alzheimer’s disease with PACAP 27 plus b-F1
ATPase antisense resulted in improved cognition of the treated animals. Thus, identification

Future Med. Chem. (2009) 1(9)

and targeting peptide efflux systems expressed
at the BBB shows renewed promise for peptide
drug design.

of efflux transporter
expression & function
Targeting signaling pathways that control drug
efflux transporter expression and function at
the BBB is another new approach to increase
brain drug levels and improve efficacy of CNS
drugs. The expectation for this strategy is that
targeting transporter regulation will decrease
transporter function and/or expression and,
thus, switch off selective, active barrier function. Such a controlled opening would provide
a ‘window-in-time’, during which drugs could
be delivered into the brain with minimal disturbance of protective barrier functions. Several
intracellular pathways that regulate BBB P-gp
have been discovered. In the following section,
we will briefly highlight two examples on how
pathways could be targeted to modulate P-gp
for opening the barrier and improving brain
drug delivery. A more detailed review on BBB
transporter regulation can be found elsewhere [9] .
Many CNS disorders are accompanied by
inflammation, which alters drug transporters
at the BBB and can therefore interfere with
drug therapy. Complex and context-dependent
regulatory networks have been found that control transporter expression and activity during
inflammation [9] . Reports suggest that P-gp
regulation is linked to the innate immune
response. In these in vitro studies, simulating
mild inflammation with LPS, TNF-a or ET-1
caused a rapid, but fully reversible, loss of P-gp
transport function in rat brain capillaries while
protein expression remained unchanged [141,142] .
This suggests inactivation of P-gp-mediated barrier function for a short period of time, during
which drugs that are P-gp substrates could be
delivered into the brain. The signaling pathway
involved binding of LPS to TLR4, with rapid
activation of TNF-R1 and ET-B receptors, followed by activation of NOS and PKC, indicating
the complexity of the mechanism. It remains to
be seen if targeting such a pathway in vivo will
increase brain levels of co-administered drugs
that are P-gp substrates.
Another clinically relevant example for targeting BBB P-gp to improve drug delivery to the
brain is epilepsy. Most of the 60 million epileptic
patients worldwide can be treated with antiepileptic drugs, but up to 40% of patients – more
than 20 million people – do not respond well to
future science group

Emerging strategies in drug delivery for overcoming the blood–brain barrier
pharmacotherapy [143–146] . This drug resistance is
at least in part due to P-gp, which, among other
transporters such as MRPs and BCRP, has been
found to be upregulated at the BBB of epilepsy
animal models and epileptic patients [147–150] .
First studies to overcome P-gp-mediated anti­
epileptic drug resistance aimed at directly inhibiting the transporter, with valspodar or elacridar
and demonstrated that this approach improved
seizure occurrence in rats [151–154] . Encouraged by
such studies, clinical trials using verapamil and
the b-blocker carvedilol to inhibit P-gp-mediated
drug resistance have recently been initiated [201] ;
the outcome of these trials remains to be seen.
Interestingly, epilepsy has a strong inflammatory
component that is reflected by elevated brain levels
of proinflammatory cytokines, prostaglandins and
the inflammatory enzyme COX-2 [155–157] . Studies
found that COX-2 is involved in seizure-induced
P-gp upregulation in epilepsy and it was shown
in a rat seizure model that indomethacin, an
un­specific COX inhibitor, and celecoxib, a specific
COX-2 inhibitor, both blocked this up-regulation
[158,159] . Studies to test if COX-2 inhibition will
increase brain levels of anti-epileptic drugs and
reduce seizures have not yet been reported. Such
proof-of-principle findings will be critical to demonstrate if this approach could be a viable therapeutic strategy to overcome transporter-mediated
drug resistance at the BBB to treat drug-resistant
epilepsy and, potentially, other CNS disorders.
Together, modulation of efflux transporters
is an interesting strategy to improve brain drug
delivery. While both approaches, direct inhibition and targeting transporter regulation, have
strengths and drawbacks, transporter inhibition
in clinical trials has so far mostly been unsuccessful and may not be a valid option. Whether
targeting transporter regulation is a viable strategy that can be carried into the clinic remains
to be seen.
Enhanced passive vascular
drug delivery
The problems associated with drug delivery to
the brain can also be overcome through the use
of methods designed to enhance passive trafficking of drugs across the BBB. These passive delivery methods can be considered as two distinct
approaches: passive trafficking of drugs between
adjacent endothelial cells after BBB disruption
(BBBD; paracellular transport); and the passive trafficking of drugs ��������������������
the endothelial cell via diffusional peptides, liposomes and
nanoparticles (transcellular transport).
future science group

The most advanced of the passive delivery systems are the methods associated with BBBD.
All of these methods disrupt BBB integrity
by breaking down the tight junctions found
between adjacent endothelial cells. If these
tight junctions are broken, drugs can easily pass
from the blood into the brain via paracellular
diffusion. Such BBBD thus facilitates the rapid
entry and distribution of CNS-nonpenetrating
drugs throughout the brain. However, there are
major limitations to the use of BBBD therapeutically. First, the disruption is transient; thus,
only a brief window of time (often only minutes) is available to deliver drugs to their therapeutic target. Second, all serum constituents,
including circulating toxicants, will also gain
access to the parenchymal compartment. And
third, some of the BBBD techniques are associated with acute sequelae, necessitating expert
administration and close medical monitoring in
human patients. Nonetheless, in lieu of other
methods for delivering drugs to the brain, use of
BBBD may find a place in the armamentarium
of clinicians treating CNS disorders.
Several methods have been recently developed
to achieve BBBD. The method most widely studied is osmotic disruption of the BBB and this has
been extensively reviewed elsewhere [124,160,161] .
Osmotic disruption involves placement of an
intra-arterial catheter in the carotid artery. A
hypertonic solution (e.g., arabinose or mannitol) is
then infused into the brain via the carotid and the
endothelial cells lining the targeted brain blood
vessels are bathed for a short period of time. The
theory behind this approach is that endothelial
cells markedly shrink in response to a hypertonic
solution and, in doing so, physically disrupt the
interendothelial cell tight junctions. The selected
neurotherapeutic is finally infused into the osmotically treated cerebrovasculature for dissemination throughout the brain. Such a procedure is
not without risk, with focal seizures reported
in a small but significant percentage of patients
[162] . To date, most clinical studies using osmotic
BBBD have been directed against treatment of
brain malignancies and have reported limited, but
nonetheless encouraging, results [160,163] .
Use of short-chain alkylglycerols and the
medium-chain fatty acid salt sodium caprate
has also been shown to disrupt the BBB in
animal models [12,164,165] . Another method for
achieving BBBD is the use of forced ultrasound
to selectively disrupt the BBB at discrete locations in the brain [166–169] . However, as these

| Review


BBB disruption
Transient disruption of the BBB
integrity by breaking down
tight junctions


Review | Stenehjem, Hartz, Bauer & Anderson
methods do not involve the use of pharmacological agents, they will not be discussed in any
more detail herein.
Several methods of pharmacologic BBBD
have been described. The best studied method
involves the use of peptide autacoids, such as
bradykinin, to disrupt the BBB [161] . Bradykinin
is a nonapeptide that, when administered systemically, transiently opens the BBB by downregulating the expression of several tight-junction proteins [170] . The kinins bind to receptors
expressed on the extracellular surface of target
cells including brain and tumor endothelial
cells [171] . However, an extremely short halflife combined with a broad range of physiologic
effects make bradykinin an unacceptable therapeutic agent. Thus, rationally designed bradykinin receptor agonists have been developed in
an effort to ameliorate the negative attributes
associated with bradykinin treatment [172] . The
best studied agonist is lobradimil (Cereport ®,
RMP-7), although additional agonists continue
to be designed [173] . Lobradimil exhibits selectivity for the bradykinin B2 receptor expressed
on brain capillary endothelial cells and not the
inducible, more broadly expressed B1 receptor  [172] . Selectivity was achieved by substituting a reduced peptide bond that serves to
protect the peptide from proteolytic cleavage
and inhibits conversion to a high-affinity B1
receptor agonist. In addition, substitution of
unnatural, chemically modified amino acids at
select positions of the nonapeptide enhance B2
receptor affinity and increase the circulating
half-life. These changes all serve to improve
BBBD while minimizing off-target effects [172] .
Several clinical trials have been conducted
using lobradimil to facilitate BBBD in combination with antineoplastic agents in the treatment of brain cancer [13,174–176] . The rationale
for these studies has been that B2 receptors are
preferentially expressed in the tumor vasculature. Thus, a ligand-activated receptor should
allow selective opening of the blood–brain
tumor barrier and facilitate the penetration
of co-administered antineoplastic agents into
the tumor [124] . The majority of clinical studies
have used carboplatin as the co-administered
agent, although loperamide and cyclosporin A
have also been tested [13] . In general, clinical
results have been discouraging. Phase I trials
have demonstrated low toxicity of lobradimil
and demonstrated the feasibility of this combinatorial therapy [177] . However, a recent pediatric Phase I trial noted unusual occurrences

Future Med. Chem. (2009) 1(9)

of early disseminated disease, prompting the
authors to speculate regarding a possible risk of
BBBD leading to a greater incidence of tumor
dissemination [174] . While the first Phase II
trial showed efficacy of combined lobradimil
(RMP‑7) and carboplatin therapy on recurrent
glioma [178] , subsequent Phase II and III trials
have not supported increased efficacy in treating
adult or pediatric brain tumors [175,176] .
Other recently discovered pharmacologic
BBB disruptors include sildenafil (Viagra®)
and vardenafil (Levitra®), inhibitors of the
phosphodiesterase type 5 (PDE5) enzyme.
Treatment of rodents with these agents resulted
in increased permeability of the blood–brain
tumor barrier and enhanced anti-tumor efficacy
when co-administered with adriamycin  [179] .
Finally, BBBD has been shown to occur in cats
after treatment with anesthetic agents such
as isoflurane [180] .

Cell-penetrating peptides (CPPs), also known
as protein-transduction domains (PTDs), represent a novel molecular mechanism for passive drug delivery to the brain. CPPs are oligocationic or amphiphilic peptide sequences of
10–30 amino acids that can transverse mammalian plasma membranes [181] . The mechanism
of CPP transport is currently believed to occur
through endocytotic uptake; however, the exact
mechanism requires further elucidation. Drugdelivery strategies employing CPPs include
chemically coupling a CPP to a therapeutic
entity. Numerous CPP families are documented
and a host of therapeutics have been delivered
to mammalian cells utilizing this technology
[182] . Nevertheless, only a few studies demonstrate CPP-mediated BBB transport of conjugated therapeutics [183] . The Syn-B CPP vectors
are the most widely characterized and in vivo
brain delivery of conjugated doxorubicin [184] ,
benzyl penicillin [185] and dalargin [186] have been
documented. However, in contrast to the Trojan
horse method­ology, CPPs currently lack specificity to target transport across the BMEC, which
may limit transport into the brain and result in
increased peripheral side effects.
Future perspective
The proof-of-principle studies described in this
review provide tantalizing support for the feasibility of rationally designing drugs capable of
both traversing the BBB and affecting neural
targets. However, hurdles remain in place that,
future science group

Emerging strategies in drug delivery for overcoming the blood–brain barrier
to date, have limited application of most of these
novel approaches to studies in animal models.
Illustrating how far we still must go is the sobering realization that l-DOPA, the best example of
a CNS drug targeting a BMEC transport system
to gain access to the brain, was found through
serendipity not rational design. And yet l-DOPA
justifiably serves to illustrate the potential that
could be realized through the rational design of
drugs targeting BBB transport systems.
What are some of the hurdles that must
be addressed in future studies? First, much
work remains regarding the biochemical
characterization of BMEC transport systems.
Promiscuous transporters, such as the Oatps,
provide an ideal target for CNS-penetrating
drugs. However, to rationally design drugs
targeting this transport system, more information regarding substrate specificity must be
collected, including iterative QSAR studies, to
better define the structural motifs recognized
by this group of transporters. Such work is not
as necessary for other CMT and RMT systems
,as most of these transporters and receptors generally accept a very limited number of substrate
structures. This lack of promiscuity proves the
major limitation for targeting many of these
other systems with novel drugs. For example,
as GLUT1 is a highly specific transporter for
d-glucose, targeting GLUT1 with novel drugs
is not possible, with the possible exception
Trojan horse conjugate drugs. Additionally,
important biochemical issues, such as identifying molecular driving forces for bidirectional
transporters such as the Oatps, remain as open
questions with important implications for vectorial drug transport. Finally, little is known
regarding the transcriptional and translational
control over transporter proteins expressed in
brain-barrier cells. Identifying the molecular
basis of control over transporter expression
would theoretically allow the rational formulation of drug cocktails designed to modulate
the expression of both influx and efflux transport systems to enhance the CNS uptake of the
targeted neurotherapeutic.
Another important issue that must be overcome for conjugate drugs is the issue of altered
target-specific activity of the conjugate. In the
design of some conjugate drugs, ester linkages
have been used to link the drug to the carrier
substrate with the rationale that the linkage will
be enzymatically broken after passage of the conjugate across the BMEC, thus releasing drug
directly to parenchymal target cells. However,
future science group

| Review

enzymatic cleavage of ester linkages may dominantly occur in the periphery, resulting in side
effects and preventing CMT-mediated transport
of the conjugate across the BBB. Moreover, CMT
of Trojan-horse conjugates may be additionally
limited by low affinity for the targeted transporter. For example, even if the affinity of novel
therapeutics is equal to endogenous substrates,
the relative plasma concentration levels could
lead to competition for transport and result in
poor conjugate delivery into the brain. Finally,
the kinetic capacity of certain transport systems
such as LAT1-mediated transport is low, limiting
utility as a CNS drug delivery system [187] .
Drug–endobiotic interactions are also a concern when targeting endogenous transport systems on the BMEC. For example, antibodies
against the transferrin receptor may upset the
tight regulation of iron homeostasis and cause
significant side effects in human clinical applications. The lack of endogenous ligands to the DTR
may present both a safety and efficacy advantage
over drug-delivery vectors targeting the insulin or
transferrin receptor. Future studies are also needed
to assess the safety and efficacy of fusion proteins
in vivo for their intended disease states and route
of administration. There are major limitations utilizing a chimeric protein approach in the interpretation of preclinical trials for regulatory approval
prior to Phase I clinical trials. These limitations
include differing BBB-targeting strategies and
species differences, which impart efficacy and
safety ramifications in delivering a therapeutic to
the brain.
Delivery of glucocerebroside to the CNS
using ApoB LDLR-fusion proteins and lentiviral
in vivo production may contribute to the future
treatment of Gaucher’s disease, as described
previously. However, targeting the CNS with
the ApoB LDLR-binding domain and lentiviral
in vivo production also has potential limitations
and biosafety concerns. The LDLR is limited
by its distribution pattern in the brain, which is
only present in a fraction of the total brain cells.
Therefore, it is unknown if therapeutic levels are
obtainable using the LDLR for CNS transport.
The potential to ellicit an immune response to
a therapeutic gene is an additional concern of
all gene-delivery systems. Lastly, utilizing lentiviral vectors for human clinical applications
raises biosafety considerations, including the
theoretical potential for oncogenesis [188] .
Additional hurdles to overcome include a
precise determination of exactly which CMT
and RMT systems are actually expressed in the


Review | Stenehjem, Hartz, Bauer & Anderson
BMEC. Most studies have focused on determining whether or not individual transporters are expressed on the BMEC. More global
approaches to determine this question are warranted. Additionally, the issue of membrane
localization is extremely important in designing
strategies targeting specific transport systems.
If the transporter is expressed in the BMEC,
is it expressed on the luminal membrane, the
abluminal membrane or both? The question is
important as blood-borne drugs must be taken
up from the blood at the luminal membrane.
If the transporter is not localized to this membrane, the drug will simply bypass the transporter and will likely not be taken up by the
BMEC. For many BMEC-expressed transport
systems, this question is not unequivocally
answered. In addition, even if the luminal versus
abluminal expression question is answered in an
animal model system such as the rat, this finding
does not necessarily translate to humans. Nor

does expression in the adult animal or human
translate to the developing neonate, even though
the vast majority of transport work is performed
solely in adults. Expression of most transport
systems is developmentally regulated with
important changes in substrate transport kinetics and even transporter preference exhibited in
the developing versus mature BBB. All of these
issues remain as important subjects for current
and future work.ary
Financial & competing interests disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a financial interest in or financial conflict with the subject matter
or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or
options, expert t­estimony, grants or patents received or
pending, or royalties.
No writing assistance was utilized in the production of
this manuscript.

Executive summary
The blood–brain barrier (BBB) provides a challenging impediment for the development of drugs targeting the CNS.
Recent molecular characterization of the BBB and identification of transporters expressed on the brain endothelial cell have revealed
novel targets for facilitating CNS drug delivery.
„„ A variety of new technologies and approaches have recently been developed that show promise in delivering neurotherapeutics across
the BBB.
„„ While many of these findings are encouraging, most studies provide proof-of-principle findings that have yet to demonstrate efficacious
therapeutic action in vivo in humans.
„„ More emphasis needs to be placed on determining whether therapeutically relevant doses of drugs can be delivered to the brain using
these novel approaches and whether the delivered drugs impact CNS targets in animal disease models.

Papers of special note have been highlighted as:
n of interest
nn of considerable interest





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