This document summarizes drug transport across the blood-brain barrier (BBB). The BBB prevents most drugs from entering the brain. Some small molecules can cross via free diffusion if they are small and nonpolar, but this applies to very few drugs. Large molecules cannot cross. However, drugs can be engineered to access carrier-mediated or receptor-mediated transport systems that naturally exist in the BBB. Injecting drugs into cerebrospinal fluid (CSF) also does not effectively deliver drugs to brain tissue due to rapid CSF turnover and limited diffusion into the brain from CSF.

1. REVIEW ARTICLE
Drug transport across the blood–brain barrier
William M Pardridge
The blood–brain barrier (BBB) prevents the brain uptake of most pharmaceuticals. This property arises from the epithelial-like tight
junctions within the brain capillary endothelium. The BBB is anatomically and functionally distinct from the blood–cerebrospinal
fluid barrier at the choroid plexus. Certain small molecule drugs may cross the BBB via lipid-mediated free diffusion, providing the
drug has a molecular weight o400 Da and forms o8 hydrogen bonds. These chemical properties are lacking in the majority of
small molecule drugs, and all large molecule drugs. Nevertheless, drugs can be reengineered for BBB transport, based on
the knowledge of the endogenous transport systems within the BBB. Small molecule drugs can be synthesized that access carrier-
mediated transport (CMT) systems within the BBB. Large molecule drugs can be reengineered with molecular Trojan horse delivery
systems to access receptor-mediated transport (RMT) systems within the BBB. Peptide and antisense radiopharmaceuticals are
made brain-penetrating with the combined use of RMT-based delivery systems and avidin–biotin technology. Knowledge on
the endogenous CMT and RMT systems expressed at the BBB enable new solutions to the problem of BBB drug transport.
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 1959–1972; doi:10.1038/jcbfm.2012.126; published online 29 August 2012
Keywords: blood–brain barrier; cerebrospinal fluid; endothelium; receptors; vascular biology
INTRODUCTION
The blood–brain barrier (BBB) prevents entry into the brain of
most drugs from the blood. The presence of the BBB makes
difficult the development of new treatments of brain diseases, or
new radiopharmaceuticals for neuroimaging of brain. All of the
products of biotechnology are large molecule drugs that do not
cross the BBB. While it is assumed that small molecules are freely
transported across the BBB, B98% of all small molecules are not
transported across the BBB.1
The lack of small molecule transport
across the BBB is illustrated by the whole body autoradiogram in
Figure 1A. The mouse was euthanized 5 minutes after the
intravenous (IV) injection of [14
C]-histamine, a small molecule of
only 111 Da. Histamine rapidly enters the postvascular space of all
organs of the body, with the exception of the brain and spinal
cord. The anatomical site of the BBB is endothelial wall of the
microvasculature in the brain.2
The density in the brain of the
microvessels is shown at the light microscopic level for rat brain in
Figure 1B, and at the electron microscopic level for the human
brain in Figure 1C. The BBB is comprised of two membranes in
series, the luminal and abluminal membranes of the brain capillary
endothelium, which are separated by about 200 nm of endothelial
cytoplasm. Owing to the presence of epithelial-like, high
resistance tight junctions within the brain capillary endothelium,
the intercellular pores that exist in the endothelial barriers in
peripheral organs are absent in the endothelial barrier in the brain.
In addition, there is minimal fluid-phase pinocytosis in brain
capillary endothelium.2
The absence of paracellular or transcellular
channels within the BBB means that molecules in the circulation
gain access to brain ISF (interstitial fluid) via only one of the two
mechanisms: (1) lipid-mediated free diffusion through the BBB or
(2) carrier- or receptor-mediated transport (RMT) through the BBB.
The BBB is the principal interface between blood and the ISF
that bathes synaptic connections within the parenchyma of the
brain. A separate barrier system in the brain is localized to the
epithelial cells of the choroid plexus, which form the blood–
cerebrospinal fluid (CSF) barrier (BCSFB). The choroid plexus is the
principal interface between the blood and CSF that bathes the
surface of the brain.
The BBB is the fundamental problem blocking progress in the
development of new therapeutics for brain disorders, or the
development of new radiopharmaceuticals for imaging brain. This
review will discuss both the principals of BBB drug transport, and
strategies for the reengineering of drugs to enable BBB transport.
The classical approaches to drug delivery to the brain, transcranial
drug delivery or small molecules, are discussed, and the limita-
tions of these strategies are reviewed. The endogenous carrier-
mediated transport (CMT) and RMT systems within the BBB are
discussed, and strategies are reviewed for the reengineering of
pharmaceuticals that penetrate the brain from blood via access of
the CMT and RMT systems within the BBB.
HISTORICAL ORIGINS OF MODERN CONCEPTS ON BRAIN DRUG
TRANSPORT
Drug distribution into CSF is frequently used as an indicator of BBB
transport. This is a misconception that stems from the lumping of
the BBB and BCSFB systems as a single brain barrier. In fact, the
BCSFB is leaky compared with the BBB, and all molecules in blood
enter the CSF at a rate inversely related to molecular weight
(MW).3
The finding of drug penetration into CSF is expected for
any molecule, and does not provide information on rates of drug
penetration across the BBB at the brain capillary endothelium.
Thus, drug distribution in CSF is not an index of BBB transport, but
rather is simply a measure of transport across the choroid plexus
at the BCSFB. The idea that CSF composition reflects BBB transport
is 100 years old, and finds its origins in Goldman’s vital dye
experiments published in 1913.4
Before 1913, it was known that
certain acidic vital dyes, such as trypan blue, are excluded from
Department of Medicine, UCLA, Los Angeles, California, USA. Correspondence: Dr WM Pardridge, Department of Medicine, UCLA, Warren Hall 13-164, 900 Veteran Avenue,
Los Angeles, CA 90024, USA.
E-mail: wpardridge@mednet.ucla.edu
Received 15 June 2012; revised 4 August 2012; accepted 9 August 2012; published online 29 August 2012
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 1959–1972
& 2012 ISCBFM All rights reserved 0271-678X/12 $32.00
www.jcbfm.com

2. entry into the brain following peripheral administration, and this
observation was explained, not within the context of a barrier
between blood and brain, but rather because the brain lacked the
‘receptors’ that bind the dye and sequester the molecule within
the tissue. Goldman injected the trypan blue into the lumbar sac,
or subdural space, of rabbits and observed vital dye staining of
spinal cord and brain tissue, respectively.4
Thus, the brain did
express vital dye binding sites. The failure to stain the brain
following peripheral administration of the dye was hypothesized
to be due to a ‘blut-gehirn schranke’ or BBB. However, in 1913, it
was believed that the pathway of nutrient flux from blood to brain
took place across the choroid plexus. The anatomical site of the
BBB was erroneously placed at the choroid plexus.4
The idea that
nutrient flux from blood to brain occurred across the capillaries
within the parenchyma of brain was not accepted in 1913.
By the 1930s, studies with brain-penetrating basic vital dyes
showed that the dye entered the brain from blood across the
walls of capillaries perfusing brain parenchyma.5
Broman6
and
Friedemann5
clearly observed that the BBB was localized to the
capillary wall in brain, and that drug entry into the CSF across the
choroid plexus was an entirely separate problem from drug
transport across the BBB. Indeed, in his 1942 review on the BBB,
Friedemann5
stated that, ‘distribution between blood and CSF is
an entirely different problem and remains outside the scope of
this review.’ Nevertheless, the idea that the BBB and BCSFB are
functionally equivalent, and that the CSF and ISF are functionally
equivalent, is perpetuated in neuroscience text books.7
These
ideas form the basis for bypassing the BBB with intrathecal drug
delivery to the brain. In 2013, or 100 years after the publication
of Goldman’s experiment defining the BBB, there are multiple
ongoing clinical trials of intrathecal delivery to the human brain of
drugs that do not cross the BBB.
BRAIN DRUG TRANSPORT AND CEREBROSPINAL FLUID
The brain has no lymphatic system. The CSF of the brain is
produced at the choroid plexi of the ventricles, moves over the
surface of the brain, and is absorbed into the general circulation
across the arachnoid villi into the superior sagittal sinus of the
venous bloodstream.8,9
In the human brain, there is about 100 to
140 mL of CSF, and this entire volume is turned over completely
every 4 to 5 hours, or 4 to 5 times per day.10
In the mouse brain,
there is about 40 mL of CSF, and the entire volume is turned over
every 2 hours, or about 12 times per day.11
Cerebrospinal fluid is a
fluid compartment in rapid equilibrium with the blood, which is
due to the rapid rate of bulk flow (convection) of CSF from brain to
blood. Conversely, drug movement into brain tissue from the CSF
flow tracts occurs via diffusion. The differential rates of convection
and diffusion create the paradox that drug injected into the CSF
distributes easily to blood and poorly to brain beyond the
ependymal surface.
Poor Drug Transport from Cerebrospinal Fluid to Brain
The poor distribution of drug into brain parenchyma is shown by
the autoradiogram of rat brain prepared 20 hours after a single
injection of [125
I]-BDNF (brain-derived neurotrophic factor) into a
lateral ventricle.12
The drug distributes only to the ependymal
surface of brain ipsilateral to the ventricular injection (Figure 2A).
From the lateral ventricle, the drug moves via bulk flow to
the third ventricle (3 V), then to the fourth ventricle, then over the
surface of the brain, where the drug undergoes exodus from the
CSF space to the venous blood. In contrast to the relative rapid
rate of bulk flow of CSF out of brain, the transport of drug from the
CSF to brain tissue is slow, and limited by diffusion. Diffusion
decreases with the square of the distance. The limited distribution
into brain from the CSF is also observed for small molecules. The
concentration of water-soluble small molecules in brain tissue at
increasing distances from the CSF surface was measured in the
primate brain following the intracerebroventricular (ICV) injection
of the drug.13
Small molecule drug concentration in the brain
decreased logarithmically with each mm of distance from the CSF
surface. There was an B10-fold decrease in drug concentration of
small molecule with each mm of distance removed from the brain
surface. In the case of a lipid soluble small molecule, which can
diffuse into brain cells, there is a 10-fold decrease in drug
concentration with each 500 mm of distance into the brain.14
The
logarithmic decrease in drug concentration in the brain removed
from the CSF surface is even steeper for a large molecule drug,
which has a lower diffusion coefficient. To achieve a therapeutic
drug level in the brain at 5 mm removed from the CSF surface, it is
necessary to inject into the CSF a dose of drug that generates at
least a 5-log increase in CSF drug concentration. This approach
Figure 1. (A) Whole body autoradiogram of a mouse euthanized
5 minutes after the intravenous injection of [14
C]-histamine.124
(B)
India ink visualization of complexity of the microvasculature of the
cortex of rat brain.125
(C) Vascular cast of the microvasculature of the
human cerebellar cortex. The distance between capillaries in brain is
about 40 mm.126
Blood–brain barrier drug delivery
WM Pardridge
1960
Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972 & 2012 ISCBFM

3. may cover drug distribution in the mouse brain, where all parts of
the brain are within 3 to 5 mm of the CSF surface. However, in the
human brain, the distance between brain and the CSF surface is
up to 50 mm, which precludes significant drug distribution into
brain parenchyma from the CSF space. A side effect of the ICV
injection of very high doses of drug is the exposure of the
ependymal surface of the brain to potentially toxic concentrations
of drug. The ICV injection of nerve growth factor in rats or
monkeys15
or basic fibroblast growth factor in rats16
causes
leptomeningeal changes with axonal sprouting and astrogliosis at
the ependymal surface of the brain.
In addition to the macrocirculation of CSF through the
ventricular system, there is a CSF microcirculation through the
Virchow-Robin compartment, which is formed by the perivascular
space around penetrating cortical precapillary arterioles, which
emanate from the pial vessels on the surface of the brain.17
However, the fluid flow via the microcirculation is quantitatively
small compared with the CSF macrocirculation. The rate of flow of
the CSF macrocirculation is 2 mL/min in the rat,8
whereas the rate
of flow of the Virchow-Robin microcirculation is only 5% of the
CSF flow rate, or 0.1 mL/min in the rat.18
Rapid Drug Transport from Cerebrospinal Fluid to Blood
Fishman and Christy19
observed nearly 50 years ago that an
intrathecal injection of drug ‘is, in effect, equivalent to no more
than a prolonged IV injection.’ The rapid movement of CSF to
blood means that drug injected into the CSF compartment will
also rapidly move to the general bloodstream. This creates the
paradox that drugs injected into the CSF may have a pharma-
cological effect within brain parenchyma, but only after transport
of the drug from CSF to blood and then to brain across the BBB
(Figure 2B). Working with barbiturates in dogs, Aird20
observed
that the anesthesia was maintained at the same dose of drug,
0.2 mg per kg per minute, whether the barbiturate was infused
by either the IV or the ICV route of administration. Drug injected
into the CSF is rapidly distributed to blood, followed by entry into
brain tissue from the blood via transport across the BBB. Drug
concentration in the dialysate of an intracerebral dialysis fiber was
measured following the ICV injection of drug; drug entry into the
blood compartment was an obligatory intermediate step between
the drug injection into the CSF and drug entry into the dialysis
fiber placed within brain parenchyma.21
A secondary effect of the
rapid movement of drug from CSF to blood is the finding of a
pharmacological response following ICV injection, owing to the
pharmacological activity of the drug in peripheral tissues. The
effect of the neuropeptide, cholecystokinin, on feeding following
ICV injection of the peptide is mediated via cholecystokinin
transfer from CSF to blood, which enables activation of chole-
cystokinin receptors in the stomach.22
Transnasal Drug Delivery to Brain
The olfactory region of the submucous space of the nose is
contiguous with the CSF flow tracts around the olfactory lobe.
Therefore, intranasal drug administration could lead to direct
delivery into the CSF providing (1) the drug is transportable across
the nasal epithelium and (2) the drug is transportable across the
arachnoid membrane separating the submucous space of the
nose and the olfactory CSF compartment.23
The properties
governing drug distribution from the nose to the olfactory CSF
are similar to the properties determining drug transport across the
BBB. A lipid soluble small molecule introduced into the nose
would be expected to have some distribution into the olfactory
CSF. However, a large molecule, or a water-soluble small molecule,
would not enter olfactory CSF from the nose in the absence of
local injury to the nasal epithelial barriers. Injury is induced in the
nose following the intranasal instillation of volumes 4100 mL/
nares in humans.24
The volume that induces local injury in the
mouse or rat nose is likely o10 mL/nares, and most studies of
intranasal drug delivery to the brain in rodents use larger volumes,
which may create nasal membrane disruption. There are species
differences between humans and rodents with respect to the part
of the nasal mucosa that corresponds to the olfactory region.
Whereas the olfactory region of the nasal epithelium constitutes
50% of the nasal mucosa in the rat or mouse, the olfactory
region is only 3% to 5% of the nasal mucosa in humans.25,26
Consequently, it is difficult to detect drug movement into CSF
following intranasal drug delivery in humans. When vitamin B12 or
melatonin was administered to humans with small volumes, for
example, 70 mL/nares, no drug distribution to CSF was observed
following intranasal drug administration.24
Convection-Enhanced Diffusion
Drug delivery to the brain via the CSF is limited by the slow
diffusion of drug from the ependymal surface of the brain
(Figure 2A). So as to replace diffusion with bulk flow, drug delivery
to the brain has been attempted with CED (convection-enhanced
diffusion). In this approach, drug is placed in a peripheral reservoir
connected to a pump, which is in line with a transcranial catheter.
In theory, drug will penetrate the brain tissue by bulk flow, which
should produce high drug levels in the brain at sites remote from
the intracerebral catheter. In practice, drug concentrations in the
brain decrease logarithmically with each mm of distance removed
from the tip of the CED catheter in the brain. A logarithmic
Figure 2. (A) Film autoradiogram of rat brain section 20 hours after
the injection of [125
I]-BDNF (brain-derived neurotrophic factor) into
the lateral ventricle (LV).12
(B) Solute distribution into brain
parenchyma from the cerebrospinal fluid (CSF) compartment is
limited by the slow rate of solute diffusion from the ependymal
surface, relative to the rapid rate of bulk flow (convection) of CSF
from the ventricles to the systemic circulation across the arachnoid
villi. (C) Logarithmic decrease in the brain concentration of glial-
derived neurotrophic factor (GDNF) in the brain of the Rhesus
monkey relative to the distance from the intracerebral catheter used
for brain drug delivery with convection-enhanced diffusion.127
Blood–brain barrier drug delivery
WM Pardridge
1961
& 2012 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972

4. decrease in the brain concentration of GDNF (glial-derived neuro-
trophic factor) is observed following the CED infusion of the
neurotrophin in the monkey brain,27
and these data are plotted in
Figure 2C. Instead of a broad distribution of the drug into brain
tissue beyond the catheter, which would be expected for a
convection or bulk flow process, there is a logarithmic decrease in
drug distribution in the brain, which is typical of a diffusion
process. It would appear that the resistance of brain tissue reduces
convection within the brain beyond the catheter tip. Similar to
intrathecal drug delivery to the brain, the CED approach requires
exposure of parts of brain proximal to the catheter to very high
drug levels, to achieve therapeutic levels of drug at sites of brain
distal to the catheter (Figure 2C).
Intrathecal or intracerebral drug delivery to the brain is limited
by diffusion, which causes logarithmic gradients of drug concen-
tration. In contrast, diffusion plays no role in the transvascular
route of drug delivery to the brain. The distance between capil-
laries in brain is about 40 mm (Figure 1C). Even a large molecule
antibody drug diffuses 40 mm within a second. Therefore, once a
drug is delivered across the BBB from blood, there is instanta-
neous equilibration of drug throughout the entire brain volume.
There are three mechanisms of trans-BBB transport of drug:
(1) lipid-mediated transport of lipid soluble small molecules;
(2) CMT transport of water-soluble small molecules that have an
affinity for an endogenous BBB CMT system; and (3) RMT transport
of large molecules that have a high affinity for an endogenous BBB
RMT system.
LIPID-MEDIATED TRANSPORT OF SMALL MOLECULES
Almost all drugs for the brain presently in clinical practice are lipid
soluble small molecules with an MW o400 Da. These drugs fit the
dual criteria for lipid-mediated free diffusion across the BBB, which
are (1) MW o400 Da threshold and (2) high lipid solubility, which
is equivalent to low hydrogen bonding. In practice, very few small
molecule drug candidates for the brain fit these dual criteria, and
do not cross the BBB. Of 46,000 drugs in the Comprehensive
Medicinal Chemistry database, only 6% are active in the brain, and
these drugs treat only a select group of brain diseases, comprised
of affective disorders and insomnia.28
In another study, only
12% of all drugs were CNS active, but if affective disorders are
excluded, only 1% of all drugs are active in the CNS.29
CNS active
drugs form r7 hydrogen bonds with solvent water.30
These
considerations indicate that the BBB transport of a small molecule
drug candidate can be predicted based on the chemical structure
of the drug, which allows for computation of (1) MW and
(2) hydrogen bonding.
Molecular Weight Threshold
Small molecule drug permeation through lipid membranes
demonstrates an MW threshold, which is not predicted by the
classical Overton rules for solute diffusion in water.31,32
Unlike
diffusion through water, solute diffusion through lipid membranes
is dependent on the molecular volume of the solute, which is
generally proportional to solute MW or solute surface area. That is,
when the size of the drug exceeds the volume threshold, then
membrane permeation is less than predicted on the basis of the
lipid solubility of the drug. The MW threshold of BBB drug
transport of small molecules has been demonstrated previously
in studies of drug penetration into the brain.33,34
Blood–brain
barrier permeation decreases 100-fold when the size of the drug is
increased from an MW of 300 Da, which corresponds to a surface
area of 50 square angstroms, to an MW of 450 Da, which cor-
responds to a surface area of 100 square angstroms.35
The
molecular mechanism of the MW threshold phenomenon is
consistent with the observation that transient water pores form
in biological membranes, which are caused by the transient
fluctuations in the conformation of the fatty acyl side chains of
membrane lipids.36–38
Hydrogen Bonding
The lipid solubility of a drug may be determined by measurement
of the 1-octanol lipid partition coefficient. However, an important
parameter, which can be determined just from an inspection of
the drug structure, is the hydrogen bonding of the drug and
solvent water, which is 55 molar. Diamond and Wright39
reviewed
the rules for determination of the number (N) of hydrogen bonds
a solute forms in water, for example, N ¼ 2 for each hydroxyl, N ¼ 3
for each primary amine, and N ¼ 4 for each amide group. Stein40
observed that there is a 10-fold decrease in membrane per-
meation with each pair of hydrogen bonds on the solute, and this
rule has been generally confirmed in studies of in vivo BBB transport
of steroid hormones41
and oligopeptides.42
Consideration of the
rules for hydrogen bonding show that even a simple dipeptide,
glycylglycine, which forms eight hydrogen bonds with water,
would have a very low BBB permeation. Conversely, if the
glycylglycine was cyclized, the number of hydrogen bonds of the
cyclic glycylglycine would be reduced to 4, and BBB transport
would be predicted to increase by 100-fold for the cyclic form
of the glycylglycine dipeptide, because glycine has no polar
side chains.
When confronted with the problem of a water-soluble drug that
does not penetrate the BBB, the medicinal chemist is asked to fix
the problem by lipidizing the drug. This can be done by blocking
Table 1. BBB carrier-mediated transporters
Carrier Substrate Genea
product Kmb
(uM) Vmax b,c
(nmol/
min/g)
Ccapd
(pmol/
mgp)
Vmax:Cape
(molecules
per second)
Glucose D-glucose GLUT1
(SLC2A1)
11,000±1,400 1,420±140 139±46 600
Monocarboxylic
acid
L-lactic acid MCT1
(SLC16A1)
1,800±600 91±35 2.3±0.8 2,300
Neutral amino
acid
L-phenyl-
alanine
LAT1 (SLC7A5) 26±6 22±4 0.43±0.09 3,000
Basic amino acid L-arginine CAT1
(SLC7A1)
40±24 5±3 1.1±0.2 270
Purine nucleoside Adenosine CNT2
(SLC28A2)
25±3 0.75±0.08 o0.14 ND
BBB, blood–brain barrier. a
Solute carrier (SLC) group of transporter genes. b
From Pardridge.47 c
Per gram wet brain in the rat. d
From Uchida et al.48
Data
reported per mg isolated human brain capillary protein. e
Vmax/Ccap ratio is the transporter turnover number, which is the number of substrate molecules
transported per second by a single transporter molecule, and is based on 0.14 mg capillary protein per gram wet brain; it is assumed that 50% of the total
capillary transporter is expressed on the endothelial luminal membrane; nd ¼ not determined.
Blood–brain barrier drug delivery
WM Pardridge
1962
Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972 & 2012 ISCBFM

5. hydrogen bond forming functional groups on the drug. For example,
when the two hydroxyl groups of morphine are acetylated, to
form heroin, the BBB penetration rate increases 100-fold.43
However, apart from the morphine/heroin example, there have
been few successes in developing new drugs for human brain
disorders by lipidization of a water-soluble drug. This is because
the hydrogen blocking functional groups are rapidly hydrolyzed
in vivo. If the hydrogen bond is blocked with a noncleavable group,
then drug activity may be reduced. An alternative approach to the
medicinal chemical modification of non-BBB-penetrating drugs is
to focus on the endogenous CMT systems within the BBB. Instead
of blocking hydrogen bonds, medicinal chemistry can be used to
modify the structure of the drug to increase drug affinity for one
of several endogenous carrier-mediated BBB transporters.
CARRIER-MEDIATED TRANSPORT OF SMALL MOLECULES
Discovery of Blood–Brain Barrier Carrier-Mediated Transport
Systems
Physiology of blood–brain barrier carrier-mediated transport. The
first evidence of saturable or CMT of a solute across the BBB was
demonstrated by Crone44
for D-glucose using the indicator dilution
technique, which is a venous sampling-carotid artery injection
method. Subsequently, the Brain Uptake Index technique, a tissue
sampling-carotid artery injection method developed by Oldendorf45
,
was used to characterize the saturable kinetics of BBB transport of
multiple water-soluble metabolic substrates. The Michelis–Menten
kinetics of BBB CMT for the major metabolic substrates were
determined,46
and this analysis showed a wide range of affinity
(Km) and maximal transport activity (Vmax) for the BBB nutrient
transporters (Table 1).
Molecular biology of blood–brain barrier carrier-mediated transport.
The methods of molecular biology were employed to identify the
principal gene product responsible for BBB transport of a given
class of metabolic substrates. The BBB glucose carrier, which also
transports other hexoses, including mannose, galactose, deox-
yglucose, or 3-0-methyl glucose,49
is the glucose transporter type
1 or GLUT1.50
The BBB phenylalanine carrier, which also transports
over 10 other large neutral amino acids,51
and to a lesser extent
small neutral amino acids, was identified as the large neutral
amino-acid transporter type 1 or LAT1.52
The BBB arginine carrier,
which also transports other cationic amino acids (lysine,
ornithine),45
was shown to be the cationic amino-acid trans-
porter type 1 or CAT1.53
The BBB lactate carrier, which also
transports other monocarboxylic acids (pyruvate, and the ketone
bodies, acetoacetate, and b-hydroxybutryyrate),54
was identified
as the monocarboxylic acid transporter type 1 or MCT1.55
The BBB
adenosine transporter, which also transports other purine nucle-
osides (guanosine, inosine), as well as the pyrimidine nucleoside,
uridine,56
is the concentrative nucleoside transporter type 2 or
CNT2 in the rat.57
Quantitation of blood–brain barrier transporter protein expression.
Terasaki and colleagues58
determined the mass of transporters in
isolated brain capillaries using the methods of LC–MS (liquid
chromatography mass spectrometry). Capillaries were isolated
from brain followed by digestion with trypsin. The tryptic peptides
were separated and identified by LC–MS. Transporter-specific
peptides were identified and quantitated by solid-phase synthesis
of synthetic peptide standards. These peptide standards, which
enable the quantification of the transporter expression, could be
synthesized based on the known amino-acid sequences of the
cloned transporters, as well as standard programs for predicting
the sequence of tryptic peptides based on known amino-acid
sequence of the target protein. The transporter concentrations in
isolated human brain capillaries, Ccap, are listed in Table 2 for
human GLUT1, MCT1, LAT1, and CAT1, and are compared with the
transporter Vmax values determined in vivo in the rat. The
comparison between rat Vmax values and human Ccap values was
made, because the Ccap values are generally comparable for
human and rodent brain capillaries.48
Transporter turnover number. The Vmax values are expressed per
gram wet brain, whereas the Ccap values are expressed per mg of
brain capillary protein. It is estimated that there is 0.17 mg
capillary protein per gram brain, since there is 100 mg protein per
gram brain, and the intracellular water volume of the brain
capillary endothelial space, 1 mL/g,61
is 1/700 parts of the total
water volume of brain. Based on this conversion, and assuming
50% of the capillary transporters are expressed on the luminal
endothelial membrane, the Vmax/Ccap ratio can be computed,
which is the transporter turnover number, or molecules of
substrate transported by the carrier per second (Table 1). The
estimated turnover numbers for BBB CMT systems range from 270
to 3,000 molecules/s. These calculations are approximations given
the divergent in vitro and in vivo methodologies that were
performed. Nevertheless, the estimated turnover rates approx-
imate the turnover rate for other transporters, for example, the
turnover number for the sucrose transporter (SUT1) is 500 mole-
cules/s.62
To design drugs that access the CMT systems listed in
Table 1, it is necessary to define the exact cellular location of the
transporter at the brain microvasculature. A transporter identified
in an isolated brain capillary preparation could be selectively
expressed on the capillary endothelium, on the capillary pericyte,
on smooth muscle cells of precapillary arterioles, or even on
astrocyte endfeet, which remain adhered to the basement
membrane of the isolated capillary. Following localization of the
transporter to the capillary endothelium, it is necessary to
determine whether the transporter is expressed on the luminal
Table 2. Reengineering of protein drugs for BBB penetration as IgG Trojan horse fusion proteins
Therapeutic class Protein therapeutic Target brain disease Animal models
Lysosomal enzymes Iduronidase (IDUA) MPS-I (Hurler’s syndrome) Rhesus monkey, Hurler mouse
Iduronate 2-sulfatase (IDS) MPS-II (Hunter’s syndrome) Rhesus monkey
Decoy receptors Type II TNFa decoy receptor (TNFR) Stroke neurodegeneration Rhesus monkey, Mouse PD, Mouse stroke
Neurotrophins Erythropoietin (EPO) Stroke Neurodegeneration Rhesus monkey, Mouse PD
Glial-derived neurotrophic factor (GDNF) Neurodegeneration Stroke Rhesus monkey, Mouse PD
Therapeutic antibodies Antiamyloid antibody (AAA) Alzheimer’s disease Rhesus monkey, Mouse AD
BBB, blood–brain barrier.
Primary references are given in Pardridge and Boado59
and Sumbria et al.60
Blood–brain barrier drug delivery
WM Pardridge
1963
& 2012 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972

6. membrane, the abluminal membrane, or both. The cellular and
subcellular expression of a BBB transporter is illustrated in the case
of GLUT1 expression at the BBB.
Expression of GLUT1 in Brain Is Localized to the Blood–Brain
Barrier
There are over 10 members of the sodium-independent GLUT
glucose transporter gene family, and the first transporter identi-
fied, GLUT1, was cloned from human HepG2 liver cells using an
antibody to the human erythrocyte glucose transporter.63
Cloning
of the cDNA allowed for prediction of the amino-acid sequence
and a two-dimensional representation of the transporter struc-
ture,63
which is shown in Figure 3A for the GLUT1 glucose
transporter cloned from a bovine BBB cDNA library.64
Shortly after
cloning from human liver cells, the same GLUT1 glucose trans-
porter cDNA was cloned from a rat brain cDNA library, and the
transporter was originally known as the brain-type glucose
transporter.65
The GLUT1 glucose transporter was observed to
be enriched at the brain microvasculature.66
However, studies of
GLUT1 glucose transporter mRNA expression in the brain with the
capillary depletion method showed that the GLUT1 glucose
transporter was expressed in the brain only at the BBB, with little,
if any, detection of GLUT1 transcript in mRNA devoid of capillary-
derived mRNA.67
The GLUT1 Northern blot for capillary-depleted
brain is illustrated in Figure 3B. The 2.9-kb GLUT1 glucose
transporter mRNA was detected in Northern blotting of non-
fractionated RNA from the brain (lane 1, left panel, Figure 3B).
However, the GLUT1 glucose transporter mRNA was 495%
removed with the capillary depletion method, which had no
effect on the abundance of a common transcript, the 2.0-kb actin
mRNA (Figure 3B). This finding indicated that GLUT1-mediated
glucose transport at the BBB, but not at brain cells. The selective
expression of the GLUT1 isoform at the BBB in the brain was
subsequently confirmed with in situ hybridization, quantitative
Western blotting,50
and quantitative immunogold electron micro-
scopy.68
The GLUT1 glucose transporter is also expressed in other
barrier structures in the brain, including the basolateral membrane
of choroid plexus epithelium, and the apical membrane of certain
regions of the ependymal epithelium.69
The localization of the immunoreactive GLUT1 glucose trans-
porter at the human BBB was demonstrated with electron micro-
scopy using an antibody directed against the human erythrocyte
GLUT1 glucose transporter and a secondary antibody conjugated
with 10 nm gold particles.70
GLUT1 glucose transporters are
observed on the endothelial plasma membranes, but not within
the neuropil of human brain (Figure 3C). The expression of the
GLUT1 glucose transporter on both luminal and abluminal mem-
branes at the human BBB illustrates how these two endothelial
membranes operate in series to regulate BBB transport (Figure 3C).
GLUT1 is also detected on the plasma membrane of the human
erythrocyte (Figure 3C). The abundance of GLUT1 within the
human erythrocyte plasma membrane is said to be highest of all
transporters. Yet, the abundance of GLUT1 glucose transporter,
expressed as transporter molecules per micron of membrane length,
is 2.6-fold higher on the luminal membrane of the human brain
capillary endothelium relative to the human red cell membrane.70
The abundance of the GLUT1 glucose transporter, Ccap, at the
microvasculature of human brain is high, relative to other
transporters, 139±46 pmol/mg protein, as determined by LC–
MS (Table 1). The Ccap value determined with LC–MS conforms to
a previous estimate of the Ccap of GLUT1, 102±25 pmol/mg
protein, determined with a cytochalasin B radioreceptor assay in
isolated brain capillaries.71
The Ccap of the GLUT1 glucose
transporter is nearly 100-fold higher than the Ccap for other
transporters (Table 1), which is attributed to the sole derivation of
Figure 3. (A) Predicted secondary structure of the bovine blood–brain barrier (BBB) GLUT1 glucose transporter is shown, which is formed by
12 transmembrane domains, a glycosylated extracellular loop between transmembrane domains 1 and 2, and intracellular amino and carboxyl
termini.128
(B) Northern blot of mRNA derived from either total rabbit brain (lane 1) or capillary-depleted rabbit brain (lane 2) for either the
GLUT1 glucose transporter or actin.71
(C) Electron microscopic immunogold study of human brain with a primary antiserum against the
purified human erythrocyte glucose transporter, and a secondary antibody conjugated with 10 nm gold particles, shows abundant expression
of immunoreactive GLUT1 glucose transporter on the luminal (L) and abluminal (AL) membranes of the capillary endothelium in human
brain.70
Blood–brain barrier drug delivery
WM Pardridge
1964
Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972 & 2012 ISCBFM

7. energy in the brain from the combustion of glucose. Cerebral rates
of glycolysis, 500 to 1,000 nmol per minute per gram, are over 100-
fold greater than rates of amino-acid incorporation into brain
proteins.46
Glucose supply in the brain is mediated solely via the
activity of the BBB GLUT1 glucose transporter.
Drug Transport into Brain via Blood–Brain Barrier Carrier-Mediated
Transporters
The characterization of the BBB GLUT1 glucose transporter
exemplifies the extensive knowledge base on the molecular
characteristics of a given BBB CMT system, and these systems have
been recently cataloged with respect to the transporter abun-
dance at the BBB.48
The cDNAs of almost all of the BBB CMT
systems are available, and cell lines can be stably transfected with
the transporter gene to enable high-throughput screening of
drugs that have an acceptable affinity for a given BBB transporter.
This knowledge base is not being exploited by the pharmaceutical
industry for the discovery of drugs that cross the BBB on a given
CMT system. However, there are historical examples of drugs that
have an unexpected affinity for a BBB CMT system that may, or
may not, be predicted on the basis of the drug structure. For
example, L-DOPA is an effective treatment for PD (Parkinson’s
disease), because this dopamine precursor is a large neutral amino
acid that is transported across the BBB via the LAT1 neutral amino-
acid carrier.51
L-DOPA is a straightforward example of a large neutral
amino-acid drug that would be predicted to have an affinity for
LAT1. However, there are examples of other drugs that have an
affinity for LAT1 that are unexpected. Gabapentin is a water-soluble
drug that is active in the CNS because the drug crosses the BBB on
the large neutral amino-acid transporter,72
which is LAT1. The
affinity of gabapentin for LAT1 is unexpected, because gabapentin
is a g-amino acid, and LAT1 has an affinity only for a-amino acids.
However, gabapentin is a cyclic form of a g-amino acid, which
places the amino and carboxyl groups in a conformation that
mimics a-amino acids. Paraquat is N,N0
-dimethyl-4,40
-bipyridinium, a
widely used, highly water-soluble herbicide, which should not cross
the BBB. However, paraquat is neurotoxic, which suggests that this
double quaternary ammonium salt is BBB penetrating. Paraquat
crosses the BBB via a process that is inhibited by valine, a large
neutral amino acid,73
which suggests that paraquat is a substrate
for LAT1. Melphalan is a polar alkylating agent used to treat brain
cancer. Melphalan, or phenylalanine mustard, crosses the BBB via
transport on the BBB large neutral amino-acid carrier.74
L-DOPA, gabapentin, paraquat, and melphalan are examples of
BBB delivery via LAT1 of drugs that have structures that mimic the
endogenous substrate, neutral amino acids. An alternative
approach is to conjugate a drug, which normally does not cross
the BBB, with a CMT substrate, for example, glucose, that does
cross the BBB. This was attempted with oligopeptides, which were
conjugated with glucose. However, the glucose moiety of the
glycopeptide no longer was transported via GLUT1.75
Instead, the
structure of the CMT substrate must be retained following conju-
gation of the nontransportable drug. This is possible with ingenious
uses of medicinal chemistry as applied to BBB structure–transport
relationships. A novel example is the case where a nontranspor-
table drug is coupled to the thiol group of L-cysteine.76
Cysteine is
a small neutral amino acid, which has a low affinity for LAT1.45
However, when the drug is conjugated to the thiol group of
cysteine, the small neutral amino acid is converted into a large
neutral amino acid, and significant BBB transport via LAT1 was
observed.76
This case shows that a CMT substrate can be used as a
drug carrier, providing the original structural characteristic of the
substrate class for the targeted CMT system is retained.
Active Efflux Transporters and Enzymatic Blood–Brain Barrier
The active efflux transporters (AET) serve to mediate the
asymmetric efflux of metabolites and drugs from the brain to
blood. The classical AET system within the BBB is p-glycoprotein,
which is a product of the MDR (multidrug resistance) gene, MDR1,
also named the ABC (ATP-binding cassette) subfamily B member 1
(ABCB1). Apart from p-glycoprotein, there are numerous other
members of the ABC gene family of transporters that are
expressed at the BBB, and these have been quantified by Terasaki
and coworkers.48
The AET systems may work in concert with
enzymes expressed within the BBB, such as P450 enzymes.77
Both
the AET systems, and the enzymatic barriers are subject to
modulation. Blood–brain barrier p-glycoprotein activity is sup-
pressed by vascular endothelial growth factor,78
and the CYP1A1
and CYP1B1 P450 enzymes are upregulated by environmental
toxins, such as dioxin derivatives, which activate the aryl
hydrocarbon receptor at the BBB.79
In addition to environmental
toxins, or growth factors, BBB permeability may be altered by
disease states, including aging.80
RECEPTOR-MEDIATED TRANSPORT OF PROTEIN
THERAPEUTICS
Discovery of Blood–Brain Barrier Receptor-Mediated Transport
Systems
Insulin is present in the brain, but there is no insulin mRNA in the
brain.81,82
Therefore, insulin in the brain is of peripheral origin and
derived from the blood. Insulin in the blood is transported across
the human BBB via RMT on the endogenous BBB insulin
receptor.83
Similarly, transferrin (Tf) in blood is transported via
RMT across the BBB on the endogenous human BBB Tf receptor
(TfR).84
There are separate BBB receptors for the insulin-like
growth factors85
and leptin.86
The BBB peptide receptors on either
the animal or human BBB were identified using isolated brain
capillaries and radioreceptor methodology. The brain capillaries
are purified from the brain with a mechanical homogenization
procedure and capillaries freshly isolated from bovine brain are
shown in Figure 4A. The BBB peptide receptors may mediate
functionally different processes, including (1) transcytosis of ligand
from blood to brain; (2) reverse transcytosis from brain to blood; or
(3) only endocytosis into the brain capillary endothelium without
net transport across the endothelial cell (Figure 4B). Insulin RMT
across the BBB from blood to brain was demonstrated with carotid
artery infusion and thaw mount autoradiography of the brain.87
Transferrin transport from the blood to brain was similarly
shown by internal carotid artery infusion and autoradiography.88
Transferrin efflux from the brain to blood was demonstrated with
the Brain Efflux Index method,89
as applied to large molecules.90
IgG molecules are asymmetrically transcytosed only in the brain to
blood direction via a Fc receptor (FcR) on the BBB.91
Confocal
microscopy of brain shows the principal FcR on the brain capillary
is the neonatal FcR or FcRn.92
The BBB FcR does not mediate the
transport of IgG molecules in the blood to brain direction. Some
BBB receptors do not mediate transcytosis across the endothelial
barrier, but only mediate endocytosis into the endothelial cell.
Acetylated LDL (low-density lipoprotein) is a ligand for the
scavenger receptor (SR) on the BBB, and this system mediates
the endocytosis of acetyl LDL from blood, but not the net
transcytosis across the BBB into the brain.93
Scavenger receptor-
related receptors, such as the LDL-related protein receptor (LRP)
type 1, similarly mediate only endocytosis of ligands into cells, and
not transcytosis.94
Proteins targeting LRP1 are taken up by brain at
reduced rates compared with proteins that target the trans-
cytosing receptor systems. The brain uptake of melanotransferrin
(p97) or angiopep-2, ligands that target LRP1, is only 0.2% to 0.3%
of injected dose (ID)/g in the mouse,95,96
which is 10-fold lower
than the brain uptake of a monoclonal antibody (MAb) that
targets the BBB TfR in the mouse.97
The BBB TfR mediates reverse transcytosis of apotransferrin in
the brain to blood direction,90
in addition to influx of holo-
transferrin from blood to brain.88
The reverse transcytosis is
Blood–brain barrier drug delivery
WM Pardridge
1965
& 2012 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972

8. enabled, because the TfR is also expressed on the abluminal
membrane of the capillary endothelium in brain. In one study, the
abluminal TfR was not detected with preembedding immune
labeling methods.98
However, abluminal receptors on the
endothelium in the brain cannot be detected with preem-
bedding methods.99
Using confocal microscopy of isolated rat
brain capillaries, and the OX26 rat MAb against the rat TfR, it was
possible to identify the TfR on both luminal and abluminal
membranes of the brain capillary endothelium (Figure 4C).100
The
pathway of transcytosis through the BBB via the TfR was examined
with electron microscopy. The OX26 TfRMAb was conjugated with
5 nm gold and infused in the carotid artery of rats for 10 minutes
followed by saline clearance of the brain vasculature and
perfusion fixation with glutaraldehyde.101
At the light
microscopic level, the TfRMAb entrapped within the capillary
endothelium was detected with a silver enhancement technique
as shown in Figure 4D. There is no visible reaction product in the
brain extravascular space (Figure 4D), owing to the differences in
intraendothelial and postendothelial volumes in the brain. The
intraendothelial volume is 1 mL/g brain and the postendothelial
volume is nearly 3-log orders higher, 700 mL/g brain. Therefore, as
the TfRMAb exits the intraendothelial volume, the antibody
concentration is diluted 700-fold, and the postvascular antibody
in the brain cannot be detected with immune labeling methods.
Figure 4. (A) Light micrograph of freshly isolated bovine brain capillaries stained with trypan blue, which stains nuclei blue and luminal red
blood cells yellow. The capillaries are isolated free of adjoining brain tissue. (B) Schematic diagram of different brain endothelial receptors,
including the insulin receptor, the transferrin (Tf) receptor, the scavenger receptor (SR), which mediates only the endocytosis from blood into
the endothelial compartment for ligands such as acetylated low density lipoprotein; and the neonatal Fc receptor (FcRn), which mediates the
asymmetric transcytosis of IgG molecules selectively from brain to blood, but not blood to brain. (C) Confocal microscopy of isolated rat brain
capillaries showing the expression of the Tf receptor on both luminal (L) and abluminal (AL) membranes of the endothelium.100
(D) Light
micrograph of rat brain removed after a 10-minute carotid artery infusion of a conjugate of 5 nm gold and the mouse OX26 monoclonal
antibody (MAb) against the rat TfR. The staining of the capillary compartment represents TfRMAb present in the intraendothelial
compartment of the brain.101
(E) Electron microscopy of rat brain after perfusion with the conjugate of gold and TfRMAb shows the MAb
concentrated in intraendothelial vesicles (intracellular arrow), as well as MAb molecules undergoing exocytosis from the endothelial
compartment to the brain interstitial space (extracellular arrow).101
Blood–brain barrier drug delivery
WM Pardridge
1966
Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972 & 2012 ISCBFM

9. The rapid postvascular distribution into the brain via transport on
the BBB TfR was demonstrated with the capillary depletion
method and autoradiography.88
At the electron microscopic level,
the OX26 TfRMAb is detected in endosomes within the capillary
endothelium (intracellular arrow, Figure 4E), and antibody is
observed exocytosing into the ISF on the brain side of the BBB
(extracellular arrow, Figure 4E).
Blood–Brain Barrier Penetrating Genetically Engineered IgG Fusion
Proteins
The presence of endogenous RMT systems within the BBB, such as
the insulin receptor or the TfR, enables the reengineering of
recombinant proteins as BBB-penetrating pharmaceuticals with
the use of molecular Trojan horse (MTH) technology.59
An MTH is
an endogenous peptide, or a peptidomimetic MAb, which traverses
the BBB on a specific RMT system. Insulin, per se, as an MTH is not
preferred, since insulin causes hypoglycemia. Tf, per se, is not a
preferred MTH, because the plasma concentration of endogenous
Tf, 25 mmol/L, is so high that the Tf-binding site on the BBB TfR is
fully saturated by endogenous Tf. Alternatively, RMT-specific MTHs
may be peptidomimetic MAbs that bind exofacial epitopes on the
BBB receptor, which are spatially removed from the endogenous
ligand binding site. Once bound to the RMT system on the BBB,
the MTH undergoes RMT across the BBB (Figure 4). An MAb
against the human insulin receptor, designated the HIRMAb,
undergoes rapid transport across the BBB of the Rhesus monkey
in vivo, and the brain uptake is 2% to 3% of ID/brain.102
The
HIRMAb crossreacts with the insulin receptor of Old World primates,
but does not react with the insulin receptor of New World primates,
such as the squirrel monkey, or lower animals such as rodents. The
murine HIRMAb has been genetically engineered as either a
chimeric or humanized antibody.103
There is no known MAb
against the mouse insulin receptor that can be used as a BBB MTH.
For BBB delivery in the mouse, a genetically engineered chimeric
MAb against the mouse TfR, designated the cTfRMAb, has been
produced.104
With the genes encoding the heavy chain and the
light chain of either the engineered HIRMAb or the cTfRMAb, IgG
fusion proteins may be genetically engineered as bifunctional
molecules, which both cross the BBB via RMT and exert pharm-
acological effects in brain, following transport into the brain.
An IgG-type MTH is also preferred for genetic engineering of
MTH fusion proteins, because the protein therapeutic can be fused
to the carboxyl terminus of the heavy chain of the IgG, as shown
in Figure 5A. It is essential that the bifunctionality of the IgG fusion
protein be retained, and that the ‘head’ of the molecule retains
high affinity (low nmol/L KD) binding to the BBB receptor, and that
the ‘tail’ of the IgG fusion protein retains the pharmacological
activity of the original protein therapeutic. If the protein
therapeutic is a neurotrophin, there must be retention of high
affinity binding of the IgG-neurotrophin fusion protein to the
cognate neurotrophin receptor in brain. If an IgG-decoy receptor
fusion protein is engineered, there must be retention of high
affinity binding to the target cytokine in the brain. If an IgG-
lysosomal enzyme fusion protein is produced, the enzyme activity
of the IgG-enzyme fusion protein must be comparable to the
native enzyme. If the therapeutic molecule is also an antibody,
then the problem is the genetic engineering of a bispecific
antibody, where there is retention of high affinity binding for both
the BBB receptor and the target antigen. This was accomplished
by fusion of an ScFv (single chain Fv) form of the therapeutic
antibody to the carboxyl terminus of the heavy chain of the IgG.105
This molecular design places the monomeric ScFv antibody in a
dimeric configuration, which restores high avidity binding to the
target antigen of a bivalent therapeutic antibody.
If the high affinity for the target BBB receptor is retained
following engineering of the IgG fusion protein, then the rate of
Figure 5. (A) Structure of an IgG fusion protein formed by fusion of a therapeutic protein to the carboxyl terminus of the heavy chain of a
blood–brain barrier (BBB) targeting monoclonal antibody (MAb). (B) Brain uptake in the mouse, expressed as % injected dose (ID)/g brain, is
shown for diazepam,110
a cTfRMAb–EPO fusion protein,59
a cTfRMAb–GDNF fusion protein,59
a cTfRMAb–TNFR fusion protein,59
and the
cationic import peptide, tat.129
(C) Brain uptake in the Rhesus monkey is expressed as % ID/100 g brain, since the size of the primate brain is
100 g. Brain uptake is shown for fallypride,111
a HIRMAb–TNFR fusion protein,109
a HIRMAb–EPO fusion protein,107
the TNFR alone,109
EPO
alone,107
and a human IgG1k isotype control antibody (hIgG1k), which is confined to the vascular compartment.108
(D) Film autoradiogram of
Rhesus monkey brain removed 2 hours after the intravenous (IV) injection of [125
I]-HIRMAb–IDUA fusion protein, showing global distribution
in brain with higher uptake in gray matter relative to white matter.130
Blood–brain barrier drug delivery
WM Pardridge
1967
& 2012 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972

10. transport of the fusion protein across the BBB will be comparable
to the rate of transport of the MTH without a fusion partner. For
the mouse, several cTfRMAb fusion proteins were engineered,
expressed, and tested for in vivo brain uptake activity. The brain
uptake of the cTfRMAb fusion protein of EPO (erythropoietin),
GDNF, or the type II TNF-a (tumor necrosis factor-a) decoy
receptor (TNFR), is high, 2% to 3% ID/g brain at 60 minutes after an
IV injection (Figure 5B). In contrast, the brain uptake of a cationic
import peptide, tat, is low, 0.1% ID/g (Figure 5B), and this low level
of brain uptake is comparable to the brain uptake of an IgG that
does not cross the BBB in the mouse.106
Without fusion to the
MTH, there is no BBB transport of the EPO alone,107
the GDNF
alone,108
or the TNFR alone.109
The brain uptake of the cTfRMAb
fusion proteins in the mouse is comparable to the brain uptake of
a lipid soluble small molecule such as diazepam (Figure 5B), which
is 1.8% ID/g at 10 minutes after an IV injection.110
Therefore,
the brain uptake of the MTH fusion proteins in the mouse is
comparable to the brain uptake of lipid soluble small molecules.
Similar findings are made in the primate. In the Rhesus monkey,
the brain uptake of the HIRMAb fusion proteins is 2% to 3% ID/
100 g brain (Figure 5C), which is comparable to the peak brain
uptake in the Rhesus monkey of fallypride, a lipid soluble small
molecule.111
The brain uptake in the Rhesus monkey is expressed
per 100 g brain, because the weight of the brain in this species is
100 g. The brain uptake in the Rhesus monkey of the TNFR alone,
or EPO alone, is low, about 0.2% ID/100 g brain. This level of brain
uptake does not represent a low level of BBB penetration, but
rather sequestration of the protein within the vascular com-
partment of brain. The primate brain uptake of an isotype IgG1
control antibody that does not cross the BBB is also 0.2% ID/100 g
at 60 minutes after an IV injection (Figure 5C). The IgG fusion
proteins undergo transcytosis through the BBB, and penetrate
brain parenchyma. The global distribution within the brain of the
fusion protein is shown by the brain scan of the Rhesus monkey at
2 hours after the IV injection of a fusion protein of the HIRMAb,
and the human lysosomal enzyme, iduronidase (IDUA) (Figure 5D).
The higher uptake in gray matter, as compared with white matter,
is due to the higher vascular density in gray matter. The finding of
transcytosis of the fusion proteins through the BBB and into brain
parenchyma is corroborated by the in vivo CNS pharmacological
effects of IV administration of the fusion proteins in mouse models
of brain disease, including Hurler’s syndrome, PD, Alzheimer’s
disease (AD), and stroke (Table 2).
BLOOD–BRAIN BARRIER TRANSPORT OF PEPTIDE
RADIOPHARMACEUTICALS FOR NEUROIMAGING
The number of potential peptide radiopharmaceuticals that could
be used to image brain disorders is large. However, peptide
radiopharmaceuticals for the brain are not developed, because
these large molecules do not cross the BBB. Peptide radio-
pharmaceuticals can be reengineered for BBB transport with the
combined use of MTH technology, avidin–biotin technology, and
radiopharmaceutical technology. The peptide radiopharmaceuti-
cal can be conjugated to the IgG MTH via a high affinity avidin–
biotin linker, as shown in Figure 6A. An MTH–avidin fusion protein
has been genetically engineered both for the HIRMAb, for primate
or human applications, and for the cTfRMAb, for mouse
studies.112,113
The IgG–avidin fusion protein is formulated in one
vial. The monobiotinylated peptide radiopharmaceutical is for-
mulated in a second vial. The two vials are mixed before IV
administration. Owing to the very high affinity of avidin binding of
biotin, there is instantaneous capture of the peptide radio-
pharmaceutical by the MTH (Figure 6A). The efficacy of this
approach has been demonstrated in imaging models in both
primates and rodents, using either the Ab1–40
amyloid peptide of
AD or epidermal growth factor (EGF) as the model peptide
radiopharmaceutical.
The Ab1–40
amyloid peptide is a potential peptide radio-
pharmaceutical for the neuroimaging of brain amyloid in AD, as
this peptide avidly binds amyloid plaque in sections of AD autopsy
brain.114
However, the Ab1–40
amyloid peptide alone does not
cross the BBB,113,115
and the absence of BBB transport prevents
the development of this peptide as a radiopharmaceutical for
brain imaging in AD. The [N-biotinyl]-Ab1–40
was radioiodinated
and injected alone IV in the Rhesus monkey.115
No measureable
brain uptake of the peptide was observed by brain scanning
(Figure 6B). However, in a second Rhesus monkey, the [N-biotinyl]-
Ab1–40
was first conjugated to the HIRMAb via a streptavidin linker
before IV injection of the HIRMAb/Ab complex. Now, the peptide
radiopharmaceutical rapidly penetrated the BBB in the primate
(Figure 6C). Following reengineering with the MTH technology,
the brain uptake of the Ab1–40
amyloid peptide is comparable to
the brain uptake of lipid soluble small molecule amyloid imaging
agents. The brain uptake of the [N-biotinyl]-Ab1–40
alone is very
low in the mouse, 0.1% ID/g, but the brain uptake of the
peptide increases to 2.1% ID/g following conjugation to the
Figure 6. (A) Conjugate of an IgG–avidin fusion protein and a
monobiotinylated peptide radiopharmaceutical. The biotin group
on the peptide binds to the avidin domain of the fusion protein to
form a high affinity linkage between the IgG molecular Trojan horse
and the peptide radiopharmaceutical. (B, C) Scans of Rhesus
monkey brain at 3 hours after the intravenous (IV) injection of
either [125
I, N-biotinyl]-Ab1–40
alone (B), or [125
I, N-biotinyl]-Ab1–40
conjugated to the HIRMAb (monoclonal antibody) molecular Trojan
horse via a streptavidin linker (C).115
(D) A rat brain tumor model was
produced by the intracerebral injection of human U87 glioma cells
in nude rats. Immunocytochemistry (ICC) of brain removed 16 days
after tumor implantation with an antibody to the human EGFR
shows abundant expression of the EGFR in the brain tumor.119
(E) Scan of rat brain removed 2 hours after the IV injection of [111
In-DTPA, Lys-biotinyl]–EGF conjugated to the TfRMAb mole-
cular Trojan horse via a streptavidin linker. The section used
for the film autoradiography was parallel to the section used for
the ICC study in (D).119
Blood–brain barrier drug delivery
WM Pardridge
1968
Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972 & 2012 ISCBFM

11. cTfRMAb–avidin fusion protein.113
This level of brain uptake of a
peptide radiopharmaceutical is comparable to the brain uptake of
a lipid soluble small molecule brain amyloid imaging agent, which
is 2% to 3% ID/g in the mouse.116
The complex of the HIRMAb–
avidin fusion protein and [N-biotinyl]-Ab1–40
retains high affinity
binding to the amyloid plaques in autopsy sections of AD brain.117
The [N-biotinyl]-Ab1–40
can be radiolabeled with 124-iodine for
positron emission tomography imaging of the brain. Alternatively,
the [N-biotinyl]-Ab1–40
can be conjugated with a chelator moiety
and labeled with 111-indium118
for single photon emission
computed tomography.
Epidermal growth factor is a potential peptide radiopharma-
ceutical for imaging brain cancers that overexpress the EGF
receptor (EGFR). Epidermal growth factor was monobiotinylated
and conjugated with diethylenediaminepentaacetic acid (DTPA)
and radiolabeled with 111-indium. A rat model of human brain
cancer was developed with the intracranial injection of human
U87 glioma cells in nude rats.119
At autopsy, immunocytoche-
mistry with an antibody against the human EGFR showed selective
expression of the EGFR in the brain tumor (Figure 6D). IV injection
of the [111
In-DTPA, biotinyl]-EGF alone enabled no imaging of the
brain tumor in vivo, because EGF does not cross the BBB.120
However, when the [111
In-DTPA, biotinyl]-EGF was conjugated to
the TfRMAb with a streptavidin linker, there was robust imaging
of the EGFR-positive brain cancer following IV injection of the
BBB-targeted peptide radiopharmaceutical (Figure 6E).
BLOOD–BRAIN BARRIER TRANSPORT OF ANTISENSE
RADIOPHARMACEUTICALS FOR NEUROIMAGING
Antisense radiopharmaceuticals are potential large molecule
agents for imaging in brain the gene expression of sequence
specific mRNA. However, antisense agents do not cross the BBB.121
An ideal antisense imaging agent is the peptide nucleic acid
(PNA). Peptide nucleic acids are resistant to nucleases, as
compared with phosphodiester-oligodeoxynucleotides (ODN),
and have less nonspecific binding than phosphorothioate-ODNs.
Peptide nucleic acid binding to target mRNAs does not trigger
RNase H activity as is observed with ODNs.122
To evaluate BBB-
targeted PNAs as antisense imaging agents for the brain, an
intracranial RG-2 brain cancer model in rats was developed.123
Gene expression within the tumor was assessed for glial fibrillary
acidic protein (GFAP) and caveolin-1a (CAV). The sequence of the
GFAP-targeted and CAV-targeted PNAs is shown in Figure 7A. The
amino terminus of the PNA was conjugated with DTPA for
radiolabelling with111
In (Figure 7A). The amide backbone of the
PNA contained a carboxyl terminal lysine (Lys) residue and the
e-amino group of the Lys residue was monobiotinylated
(Figure 7A). Confocal microscopy showed the intracranial brain
tumor underexpressed the GFAP protein and overexpressed the
CAV protein (Figure 7B). Northern blot analysis showed the level of
the GFAP and CAV mRNAs in the tumor paralleled the protein, as
the GFAP mRNA was underexpressed, and the CAV mRNA was
overexpressed.123
The biotin-GFAP-PNA or the biotin-CAV-PNA
was conjugated to the TfRMAb via a streptavidin linker.
Conjugation of the PNA to the MTH did not affect binding of
the PNA to the target transcript. This was demonstrated with
Northern blotting using the labeled PNA as the probe, and cloned
GFAP or CAV RNA as the target.123
Following IV injection of either
the [111
In-DTPA, biotinyl]-GFAP PNA alone, or the [111
In-DTPA,
biotinyl]-CAV PNA alone, there was no imaging of brain, as these
molecules do not cross the BBB. Following the IV injection of the
[111
In-DTPA, biotinyl]-GFAP PNA/TfRMAb conjugate, the nontumor
brain was imaged, but there was no signal over the tumor
Figure 7. (A) Structures are shown for model peptide nucleic acids (PNAs) with sequences that target either the rat glial fibrillary acidic protein
(GFAP) mRNA or the rat caveolin-1a (CAV) mRNA.123
The methionine initiation codon sequence is underlined. (B) Confocal microscopy of brain
from a rat with an intracranial RG-2 glioma is shown after double labeling with an antibody against GFAP (green channel) and an antibody
against CAV (red channel).123
(C) Scan of brain removed from a rat with an intracranial RG-2 glioma at 1 hour after the intravenous (IV)
injection of the [111
In-DTPA, Lys-biotinyl]-GFAP PNA conjugated to the TfRMAb (monoclonal antibody) via a streptavidin linkage. The image
over the tumor is of low intensity, as the RG-2 tumor does not express the GFAP mRNA.123
(D) Scan of brain removed from a rat with an
intracranial RG-2 glioma at 6 hours after the IV injection of the [111
In-DTPA, Lys-biotinyl]-CAV PNA conjugated to the TfRMAb via a streptavidin
linkage. The image over the tumor is of high intensity, as the RG-2 tumor expresses abundant CAV mRNA.123
Blood–brain barrier drug delivery
WM Pardridge
1969
& 2012 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972

12. (Figure 7C). Therefore, the in vivo brain scan with the targeted
GFAP-PNA antisense radiopharmaceutical confirmed the in vitro
finding of minimal expression of the GFAP mRNA in the glial tumor.
Following the IV injection of the [111
In-DTPA, biotinyl]–CAV PNA/
TfRMAb conjugate, the tumor showed an intense signal, owing to
overexpression of the CAV mRNA in the tumor (Figure 7D). The
in vivo brain scans (Figures 7C and 7D) of the GFAP or CAV mRNAs
with BBB-targeted antisense radiopharmaceuticals paralleled the
immune detection of GFAP or CAV proteins in autopsy sections
(Figure 7B), and the levels of the GFAP or CAV mRNAs in the
tumor.123
Peptide and antisense radiopharmaceuticals have the
potential to greatly expand the diagnostic capabilities of modern
neuroimaging. However, these molecules need to be reformulated
for BBB transport, and this is possible with the combined use of
MTH and avidin–biotin technologies.
In summary, small molecule drugs may be reengineered to
enable transport across the BBB via the CMT systems expressed in
the brain capillary endothelium. Large molecule drugs, including
recombinant proteins, and peptide and antisense radiopharma-
ceuticals may be delivered across the BBB with MTHs that target
the endogenous RMT systems expressed within the brain capillary
endothelium. The reengineering of pharmaceuticals for BBB
transport is enabled with knowledge on the endogenous CMT
and RMT systems within the BBB.
DISCLOSURE/CONFLICT OF INTEREST
The author is a consultant to ArmaGen Technologies.
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1972
Journal of Cerebral Blood Flow & Metabolism (2012), 1959 – 1972 & 2012 ISCBFM