Factors That Limit Positron Emission Tomography Imaging of P-Glycoprotein Density at the Blood-Brain Barrier

Abstract

Efflux transporters located at the blood-brain barrier, such as P-gp and BCRP, regulate the passage of many drugs in and out of the brain. Changes in the function and density of these proteins, in particular P-gp, may play a role in several neurological disorders. Several radioligands have been developed for measuring P-gp function at the blood-brain barrier of human subjects with positron emission tomography (PET). However, attempts to measure P-gp density with radiolabeled inhibitors that bind to these proteins in vivo have not thus far provided useful, quantifiable PET signals. Herein, we argue that not only the low density of transporters in the brain as a whole but also their very high density in brain capillaries act to lower the concentration of ligand in the plasma and thereby contribute to absent or low signals in PET studies of P-gp density. Our calculations, based on published data and theoretical approximations, estimate that whole brain densities of many efflux transporters at the blood-brain barrier range from 0.04 to 5.19 nM. We conclude that the moderate affinities (> 5 nM) of currently labeled inhibitors may not allow measurement of efflux transporter density at the blood-brain barrier, and inhibitors with substantially higher affinity will be needed for density imaging of P-gp and other blood-brain barrier transporters.

Full text

Factors That Limit Positron Emission Tomography Imaging of
P‑Glycoprotein Density at the Blood−Brain Barrier
Pavitra Kannan,
†,‡
Victor W. Pike,
†
Christer Halldin,
‡
Oliver Langer,
§,∥
Michael M. Gottesman,
⊥
Robert B. Innis,*
,†
and Matthew D. Hall
⊥
†
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, United
States
‡
Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden
§
Health and Environment Department, Biomedical Systems, AIT Austrian Institute of Technology GmbH, 2444 Seibersdorf, Austria
∥
Department of Clinical Pharmacology, Medical University of Vienna, 1090 Vienna, Austria
⊥
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
ABSTRACT: Efflux transporters located at the blood−brain
barrier, such as P-glycoprotein (P-gp) and breast cancer
resistance protein (BCRP), regulate the passage of many drugs
in and out of the brain. Changes in the function and density of
these proteins, in particular P-gp, may play a role in several
neurological disorders. Several radioligands have been
developed for measuring P-gp function at the blood−brain
barrier of human subjects with positron emission tomography
(PET). However, attempts to measure P-gp density with
radiolabeled inhibitors that bind to these proteins in vivo have
not thus far provided useful, quantifiable PET signals. Herein,
we argue that not only the low density of transporters in the
brain as a whole but also their very high density in brain capillaries act to lower the concentration of ligand in the plasma and
thereby contribute to absent or low signals in PET studies of P-gp density. Our calculations, based on published data and
theoretical approximations, estimate that whole brain densities of many efflux transporters at the blood−brain barrier range from
0.04 to 5.19 nM. We conclude that the moderate affinities (>5 nM) of currently labeled inhibitors may not allow measurement of
efflux transporter density at the blood−brain barrier, and inhibitors with substantially higher affinity will be needed for density
imaging of P-gp and other blood−brain barrier transporters.
KEYWORDS: positron emission tomography (PET), ABC transporters, imaging, blood−brain barrier, inhibitors, density
■INTRODUCTION
P-Glycoprotein (P-gp, encoded by ABCB1) and breast cancer
resistance protein (BCRP, encoded by ABCG2) are among the
efflux transporters that are essential to the function of the blood−
brain barrier.
1
These two transporters are apically located
(blood-facing) in the endothelial cells of brain capillaries to
prevent the entry of drugs into the brain, thereby protecting it
from exposure to toxins in the blood.
2,3
As a result, P-gp and
BCRP can also impede the entry of potential therapeutics to the
brain, and this barrier function may be exacerbated in
pathophysiological conditions. Increased function of P-gp, for
example, may contribute to drug resistance in epilepsy and may
decrease the effectiveness of treating HIV infection of the
brain.
4,5
Changes in expression (i.e., density) of these trans-
porters may also be clinically relevant because expression affects
efflux capacity. For example, loss of P-gp expression would result
in net decrease in function and, consequently, in dysfunction of
the blood−brain barrier. Because the function and density of P-
gp and BCRP in neurological disorders are not well understood
in vivo, quantifying both transporter function and density is a
significant and important challenge.
Molecular imaging techniques such as positron emission
tomography (PET) offer the potential for in vivo measurement of
function and density of protein targets. In PET, a radiotracer is
injected at a subpharmacological dose, and biomathematical
modeling is applied to acquired data to determine output
measures related to the interaction of the radiotracer with the
receptor target. In the case of functional studies, increased or
decreased uptake of a radiotracer measures protein function.
Glucose metabolism in tissue, for example, is reflected by
increased uptake of [18F]fluorodeoxyglucose.
6
Efflux transport
by P-gp or BCRP, on the other hand, is reflected by little to no
uptake of the radiolabeled substrate in tissue, and inhibition of
efflux transport results in increased substrate accumulation.
1
PET
Received: January 8, 2013
Revised: March 27, 2013
Accepted: April 18, 2013
Published: April 18, 2013
Review
pubs.acs.org/molecularpharmaceutics
© 2013 American Chemical Society 2222 dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−2229

has been successful in using radiolabeled substrates to measure P-
gp function at the blood−brain barrier for at least two reasons.
First, each molecule of P-gp can transport multiple substrate
molecules. Second, upon inhibition of transport, amplification of
the PET signal can occur by trapping some of the substrate in
cellular organelles.
7,8
In the case of density studies, the density of the target is
inferred from the binding potentiala parameter that is the
product of the concentration of binding sites (Bmax) and the
affinity of the radioligand for the protein (1/KD, where KDis the
equilibrium dissociation constant).
9
[The term radioligand
describes a specific class of compounds that reversibly or
irreversibly bind to the protein target (e.g., radiolabeled
inhibitor), whereas the term radiotracer refers to a more general
class of compounds that includes substrates and radioligands.]
Because substrates are transported quickly when in the vicinity of
a transporter,
10
they cannot be used to measure the density of
these efflux transporters by PET. Inhibitors of P-gp or of BCRP,
on the other hand, are known to directly bind to their respective
transporters
10
and might behave akin to antagonist receptor
radioligands, thereby measuring density. However, PET has been
unsuccessful at measuring P-gp density using radiolabeled
inhibitors. Unlike the signal amplification obtained by substrate
trapping, the maximal signal that can be obtained by binding is
one radiolabeled inhibitor for each transporter (assuming one
binding site per transporter). The difficulty in imaging P-gp
density is also due to two previously described phenomena. First,
many of the inhibitors cross-react with P-gp and BCRP,
11
which
confounds results obtained in P-gp knockout, Bcrp knockout, or
P-gp/Bcrp knockout mice. Second, some inhibitors of P-gp are
also substrates for BCRP.
12
At very low concentrations, high
affinity inhibitors might be transported by P-gp, but evidence for
this appears to be conflicting.
13,14
The purpose of this perspective is to highlight two other
difficulties of imaging P-gp density at the blood−brain barrier
using PET. The first is that the density of P-gp in the whole brain
has been variably reported in the literature but plays a critical role
in the affinity of the radioligand required to have a measurable
signal from in vivo PET imaging. This may be exacerbated by the
low resolution of PET imaging. The second difficulty, which has
not been discussed in the literature, is the high density of P-gp
within the local microenvironment (microcompartment) of the
capillary. We propose that the density of P-gp (and BCRP) is
high enough within the capillary compartment to substantially
affect the free concentration of radiotracer during its 1−2 s transit
through the capillaries. Here, we use the results from PET studies
of P-gp density to explain how these two factorslow binding
potential and high, localized transporter densitymay greatly
affect brain signal.
In the following sections, we assume for simplicity that: (1)
The brain signal measured in PET studies of P-gp density is
“binding to P-gp”. In reality, “brain uptake of radioactivity”
includes both parenchymal uptake (after passing the blood−
brain barrier) and radioactivity in the vascular compartment
(about 5% of total brain volume (see below)). PET studies seek
to determine the amount of radiolabeled inhibitor bound to P-gp
in the brain capillaries. However, PET lacks the resolution to
separately measure uptake in parenchyma and that in the vascular
compartment. (2) A radiolabeled inhibitor can bind to all the P-
gp in brain endothelial cells, but the majority of P-gp is localized
at the luminal membrane. While P-gp is constantly recycled on/
offthe cell surface, the majority is thought to be expressed at the
cell surface.
■RESULTS FROM PET STUDIES MEASURING P-GP
DENSITY
PET studies using high-affinity P-gp/BCRP inhibitors to
measure transporter density at the blood−brain barrier have
yielded confusing results (Table 1). According to conventional
PET studies measuring receptor density, the specific binding of a
radioligand to its receptor should be displaceable.
9
If we assume
that a radiolabeled inhibitor binds only to P-gp (i.e., is specific),
then high concentrations of unlabeled inhibitor should displace
the radioligand bound to P-gp and consequently decrease the
measured brain signal. However, brain uptake of all except one of
the tested radiolabeled inhibitors was low 30 min after injection
in wild-type rats but increased at least 150% after blockade with
high doses of nonradiolabeled inhibitors; the increase was not
due to altered peripheral metabolism after blockade.
15−19
Results
obtained from transgenic mice are also not consistent with
expectations for a P-gp binding molecule. For example, brain
uptake of the “high-affinity”P-gp inhibitor [11C]tariquidar was
low in wild-type, P-gp (mdr1a−/−/mdr1b−/−6), and Bcrp(abcg26)
knockout mice but was high in P-gp/Bcrp (mdr1a−/−/mdr1b−/−/
Table 1. Brain Concentrations of Radiolabeled P-gp and BCRP Inhibitors in Rat and Mouse Measured 30 min after Injection
radiotracer uptake in rat brain at
25 min radiotracer uptake in mouse brain at 30 min
radiolabeled inhibitor baseline blocked unit
a
wild-type P-gp knockout BCRP knockout P-gp/BCRP knockout unit reference
[11C]laniquidar 0.07 0.6 %ID/g 18
[11C]elacridar 0.05 0.15 0.05 1.0 SUV 22
0.20 1.08 SUV 0.18 0.46 0.20 1.39 SUV 16
1-[18F]fluoroelacridar 0.14 1.29 SUV 0.23 1.76 SUV 17
[18F]fluoroethylelacridar 0.08 0.18 0.08 0.70 SUV 21
[11C]tariquidar 0.02 0.04 0.02 0.75 SUV 20
0.19 0.54 SUV 0.15 0.31 0.18 1.39 SUV 15
[18F]fluoroethyltariquidar 0.07 0.14 0.10 0.60 SUV 21
[11C]-1
b
1.2 0.90 SUV 19
[11C]-2
c
0.96 1.17 0.80 1.79 SUV 23
a
%ID/g = % injected dose/gram of tissue. SUV = standardized uptake value, which is the ratio of radioactivity concentration measured in tissue at
time t to injected dose at time of injection divided by body weight. A SUV of 1.0 is the value that would be obtained throughout the body for a
hypothetical perfectly uniform distribution.
b
1 = novel P-gp inhibitor, [11C]6,7-dimethoxy-2-{3-[4-methoxy-3,4-dihydro-2Hnaphthalene-(1E)-
ylidene]-propyl}-1,2,3,4-tetrahydro-isoquinoline
c
2 = putative BCRP inhibitor, [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoqunolin-2-
yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-2-carbonylamino)benzoate
Molecular Pharmaceutics Review
dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−22292223

abcg26) triple knockout mice.
15,20
Tariquidar is a substrate for
BCRP,
12
which explains its low uptake in wild-type and P-gp
knockout mice. However, one would have expected higher
uptake in Bcrp knockout mice because P-gp is still expressed in
brain capillaries and can bind to [11C]tariquidar. Brain uptake of
other radiolabeled inhibitors followed a similar pattern in the
four strains of mice.
16,17,21,22
Although extensive work has been done on P-gp inhibitors,
only one putative BCRP inhibitor has been radiolabeled and
tested in animals. The brain uptake of this 11C-labeled compound
(chemical name [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinolin-2-yl)ethyl)phenyl)amino-carbonyl)-2-
(quinoline-2-carbonylamino)benzoate
23
) in wild-type mice was
high but was even higher in P-gp and P-gp/BCRP knockout
mice, similar to results obtained with most of the P-gp
inhibitors.
23
Among these eight reported ligands, only one inhibitor
showed a different uptake profile. The P-gp inhibitor [11C]-1
(chemical name 6,7-dimethoxy-2-{3-[4-methoxy-3,4-dihydro-
2Hnaphthalene-(1E)-ylidene]-propyl}-1,2,3,4-tetrahydro-iso-
quinoline
19
) had a high brain uptake (>1.0 SUV) in rats at
baseline conditions and a 30% reduced uptake after blockade of
transporters.
19
While these results are what one might expect
from a PET study measuring efflux transporter density, they
should be interpreted cautiously. The inhibitor [11C]-1 is
approximately 2 orders of magnitude less potent than tariquidar
and elacridar
24
and may not, therefore, have the affinity required
to specifically measure the density of P-gp at the blood−brain
barrier (see discussion below).
■TWO FACTORS AFFECT BRAIN SIGNAL IN PET
STUDIES OF P-GP DENSITY
Low Binding Potential of Efflux Transporters. Even after
blockade or genetic knockout of transporters, the raw values of
brain uptake for most of the radiolabeled inhibitors are still lower
(<1.8 standardized uptake value, SUV, Table 1) than those
typically achieved with PET radioligands that bind to a specific
receptor in the brain.
25
The fact that little or no brain signal was
observed in conditions where P-gp is expressed and its density
should be measurable suggests that either the density of P-gp in
the whole brain (Bmax) is not sufficient to allow visualization by
PET or that the affinity (1/KD) of the inhibitor is not high
enough. In other words, while an inhibitor may bind to P-gp at
the blood−brain barrier, binding in the whole brain is not
detectable by PET because the binding potential (Bmax/KD)of
the whole brain is too low. For a radioligand to have measurable
brain uptake, the Bmax should exceed the KDof the ligand [A value
of >5 for binding potential (Bmax/KD) is generally recommended
to achieve measurable brain uptake; however, the field lacks
consensus on this value]: if a binding site has nanomolar
concentrations in vivo, then the radioligand should ideally have
subnanomolar affinity.
26
What, then, is the binding potential for
P-gp and a high-affinity inhibitor (e.g., tariquidar) in the whole
brain?
Estimation of Efflux Transporter Density in the Whole Brain
(Bmax). To calculate the binding potential, an estimate of P-gp
density in the whole brain (Bmax) is required. Although the
affinities of radiolabeled inhibitors for P-gp have been
experimentally determined, the Bmax has not [reports have
been published on P-gp expression in cells of the brain
parenchyma expressing P-gp (particularly during neuroinflam-
mation) and in infiltrating lymphocytes]. However, for the
purposes of this discussion we will assume that P-gp expression in
Table 2. Calculated Values of Receptor Density for Various Efflux Transporters Expressed in Human, Monkey, and Mouse Brains
a
human monkey mouse
gene protein protein conc.
(fmol/μg) calculated Bmax
(nM) protein conc.
(fmol/μg) calculated Bmax
(nM) protein conc.
(fmol/μg) calculated Bmax
(nM)
ABC Transporters
ABCA2 2.86 0.61 NR ULQ
ABCA8 1.21 0.26 NR ULQ
ABCB1 P-gp 6.06 1.28 4.71 1.00 14.1 2.99
ABCC4 Mrp4 0.195 0.04 0.286 0.06 1.59 0.34
ABCG2 Bcrp 4.07 0.86 7.10 1.51 2.20 0.47
SLC Transporters
SLC1A3 EAAT1 24.5 5.19 NR NR
SLC2A1 GLUT1 139 29.47 129 27.35 90.0 19.08
SLC2A3 GLUT3 4.40 0.93 1.22 0.26 ULQ
SLC3A2 4F2hc 3.47 0.74 NR 16.4 3.48
SLC6A12 BGT1 3.16 0.67 NR NR
SLC7A1 CAT1 1.13 0.24 NR NR
SLC7A5 LAT1 0.431 0.09 ULQ 2.19 0.46
SLC16A1 MCT1 2.27 0.48 0.834 0.18 23.7 5.02
SLC19A1 RFC 0.763 0.16 NR NR
SLC29A1 ENT1 0.568 0.12 0.541 0.11 0.985 0.21
Receptors
INSR insulin receptor 1.09 0.23 1.52 0.32 1.16 0.25
LRP1 Lrp1 1.51 0.32 1.29 0.27 1.07 0.23
Tf R1 transferrin
receptor 2.34 0.50 NR 5.84 1.24
a
ABCG2/BCRP values are half that reported in Uchida and colleagues,
29
to account for the homodimerization of protein product to form a
functional unit. ULQ = Under limit quantification. NR = Gene not reported/examined.
Molecular Pharmaceutics Review
dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−22292224

the brain occurs in the brain capillary endothelial cells only.
Ordinarily, in vitro studies with brain homogenate are used to
determine an approximate Bmax value,
9
but the high lipophilicity
of the P-gp inhibitors (e.g., clog Ptariquidar = 6)
27
has made it
difficult to estimate this value using this method. Instead, we use
the values published recently by Terasaki and co-workers, who
measured the expression of transporters in isolated brain
capillary endothelial cells using quantitative liquid chromatog-
raphy-tandem mass spectrometry. This method allows for
measurement of protein levels, given as fmol/μg of cellular
protein in endothelial cells isolated from brain capillaries of
mice,
28
monkeys,
29
and humans
30
(summarized in Table 2).
From these studies, we know the concentration of transporters
in human brain capillary endothelial cells, but not in the whole
brain, which is necessary to calculate Bmax. Therefore, the
calculation of the concentration of a protein expressed in
capillary endothelial cells in the whole brain is critically reliant on
knowledge of the density of brain capillary endothelial cells as a
proportion of total brain tissue. This is distinct from the total
brain capillary volume, which is much higher, as it includes blood
volume. Blood volume in the brain is reported to be
approximately 5% of total brain volume,
31,32
but the capillary
volume is significantly lower. A three-dimensional reconstruction
of brain capillaries from frozen serial sections of feline temporo-
parietal Suprasylvian−Gyrus revealed an average capillary
diameter of 4.68 ±0.92 μm, total capillary length of 42.55 cm/
mm2of brain tissue, and a capillary volume of 2.8%.
33
Yet there is surprisingly little experimental data on the volume
occupied by endothelial cells in the brain. The principal values
cited come from Pardridge,
34
who stated that, “Since the brain
capillary endothelial cell volume is only 1 μL/g brain, the
endothelial volume in brain is only 0.2% of the total cell volume
in brain.”In an earlier report, Pardridge estimated that
endothelial cell volume is 0.8 μL per g of brain, given a capillary
volume of 1% of total brain volume, and that endothelial cells
occupy one-tenth of the total capillary volume (Figure 1).
35
Is there experimental support for these approximations? The
most useful data appears to come from Hicks and colleagues,
who assessed the cross-sectional area of capillary components
(basement membrane, endothelial cell, lumen, and pericyte) in
the hippocampus and frontal cortex of rats of 3, 9, and 24 months
of age.
36
Assessment of area was achieved by producing large
photographic copies of each electron microscope image, cutting
out the components, and weighing the pieces to calculate area.
The percentage of area composed of endothelial cells in the
frontal cortex was 12% and in the hippocampus was 11%
(average basement membrane 4%, lumen 80%, and pericyte 4%).
Given an experimental representative brain capillary volume of
2%, and an experimental representative endothelial cell volume
that is 10% of the total capillary volume, brain capillary
endothelial cell content is indeed estimated at 0.2% of brain
volume.
Using the numbers from previous studies, we estimate that the
Bmax of P-gp in the whole human brain is 1.3 nM (see Appendix A
for detailed calculations). The method employed by Terasaki and
colleagues to measure the expression of transporter protein did
not differentiate between functional transporter at the luminal
membrane and internalized transporter; as such, Bmax at the
luminal membrane may be slightly lower (as noted earlier,
inhibitors may bind both pools of P-gp). We have determined the
Bmax values for other brain capillary transporters in a similar
fashion (Table 2). It is notable that only two solute carrier
transporters demonstrate higher expression in human brain
endothelial cells than P-gp. They are associated with energy
supply to the brain (glucose transporter 1, Glut1, SLC2A1,
calculated brain concentration 29.5 nM) and glutamate supply
(glutamate aspartate transporter, Eaat1, SLC1A3, calculated
concentration 5.2 nM). While PET functional studies of Glut1
have been reported, and it is expressed only in brain endothelial
cells,
37
no PET binding studies exist in the literature for this
transporter.
Binding Potential (Bmax/KD) of P-gp and “High-Affinity”
Inhibitors. Using the Bmax value mentioned above for P-gp (1.3
nM) and reported KDvalues for various P-gp inhibitors, we
calculate that Bmax/KDvalues range from 0.004 to 0.470 (Table
3). These values are well below the Bmax/KDcutoffvalue of >5−
10 recommended for a useful PET radioligand.
26
Although a
similar mathematical approach was described in the literature,
previous calculations used a vascular volume of 5% rather than a
capillary density of 0.2%,
15
concluding that the Bmax/KD(i.e., the
binding potential) for tariquidar, for instance, is 15, which would
Figure 1. Diagram of cross-section from a rat brain capillary
demonstrating that endothelial cells constitute a small percentage of
the total capillary volume. The diagram was generated from electron
micrograph published by Hicks and colleagues.
36
The scale bar
represents 1 μm.
Table 3. Calculated Values of Binding Potential (Bmax/KD)
a
for Various P-gp Inhibitors
inhibitor KD
b
(nM) species used for
cell assay reference calculated Bmax/KD
for P-gp
cyclosporin A 300 hamster 42 0.004
elacridar 2.7 human 43 0.474
tariquidar 5.1 hamster 14 0.251
valspodar 80 hamster 42 0.016
zosuquidar 55 hamster 42 0.023
a
Bmax = concentration of binding sites. KD= equilibrium dissociation
constant, where affinity of the ligand for the target is defined as 1/KD.
b
The KDvalue for the P-gp inhibitor, laniquidar, has not been
reported.
Molecular Pharmaceutics Review
dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−22292225

allow for obtaining a sufficiently high in vivo signal with
radiolabeled tariquidar (subsequently corrected
38
).
There are clearly limitations to our calculations because they
are based on experimentally determined values derived from a
range of species: rabbit (brain capillary density), rat (capillary
cross sections), human (capillary transporter density and brain
protein density), and hamster (inhibitors’KDfor P-gp).
Nevertheless, it is clear that the concentration of P-gp in the
whole brain is very low (<1 nM). Given such a low Bmax for P-gp
in the whole brain, the affinity of tariquidar and other “high-
affinity”inhibitors needs to be at least an order of magnitude
higher (picomolar) to allow PET imaging. For example, based on
a calculated Bmax of 1.3 nM for P-gp in the whole brain and a
binding potential threshold of 5 for imaging, a KDof 260 pM (or
lower in value) would need to be achieved before imaging the
density of P-gp is possible.
High, Localized Transporter Density. Although conven-
tional PET studies of receptor density in the brain require brain
penetration of a radioligand,
25
PET studies of transporter density
do not, as the target (i.e., efflux transporters) is located within
capillary walls. At this location, which is exposed to the plasma
compartment, efflux transporters can have a direct effect on the
free concentration of radioligand. Because only free radioligand
can bind to a target, we speculate that this effect on drug
disposition is more likely to affect tracer concentrations of drug
than pharmacological doses.
We propose that the local density of P-gp in the endothelial
cells is so high that it transiently binds a high percentage of
radiolabeled inhibitor, thereby lowering the free concentration in
the capillary such that a negligible amount would enter the brain.
If all P-gp is concentrated in the capillary volume, then its
concentration (Bmax) in the capillary space is 40 nM. To illustrate
the effect of high, localized density on free ligand concentration,
Figure 2. The high, localized density of P-glycoprotein (P-gp) may transiently reduce the free plasma concentration of [11C]tariquidar in brain
capillaries, which may subsequently reduce the concentration of free radioligand in the brain. (A) Uptake of [11C]tariquidar, as measured by positron
emission tomography (PET), is low in brains of wild-type and Bcrp knockout, 2-fold higher in P-gp knockout mice, and 9.3-fold higher in P-gp/Bcrp
knockout mice. Modified with permission from data published by Langer and colleagues
15
(2010). Copyright 2010 Elsevier. (B) Scheme demonstrating
the potential interactions of radiolabeled inhibitor [11C]tariquidar (indicated by *) with transporters P-gp (red) and breast cancer resistance protein
(Bcrp, yellow). The numbers to the right of each image are representative values for the density (Bmax) of P-gp, free concentration of tariquidar inplasma
([TQR]FP), and free concentration of tariquidar in brain ([TQR]FB) for each mouse strain. The values were calculated assuming that the density of P-gp
is 40 000 pM in the capillaries, and that tariquidar has a concentration of 1 nM in the capillary, a KDof 5 nM, and a free fraction of 0.05 (meaning a free
concentration of tariquidar of 0.50 pM). In wild-type and Bcrp knockout mice, for every one molecule of tariquidar that is free, eight molecules are
bound to P-gp; this binding further decreases the free concentration of tariquidar in plasma to 0.06 pM. In P-gp knockout mice, the free concentration of
tariquidar is not further reduced by high-affinity binding to P-gp, although overall parenchymal uptake is still low because the inhibitor is efficiently
effluxed as a substrate of Bcrp. In P-gp/Bcrp knockout mice, the radiolabeled molecule is taken up in the brain and trapped (probably by lysosomal
trapping
7
).
Molecular Pharmaceutics Review
dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−22292226

we used the Michaelis−Menten equation to calculate the
bound−free ratio and receptor occupancy for the inhibitor
tariquidar (see Appendix B). As the concentration of free
radioligand decreases, the bound−free ratio increases to a
maximum of eight. In other words, for each free molecule of
tariquidar in the brain capillary, eight molecules are bound to P-
gp. Despite this high bound−free ratio, the receptor occupancy at
this concentration (0.001 nM) is very low (0.02%).
What would be the predicted receptor occupancy with tracer
doses of [11C]tariquidar in PET? Radioligands that are too
lipophilic are highly bound to plasma proteins, which would
dramatically reduce the free plasma concentration of radio-
ligand.
39
Like elacridar and laniquidar, tariquidar is a highly
lipophilic compound (clog P=6)
27
that is highly bound to
plasma proteins: we have observed the free plasma concentration
of [11C]tariquidar to be 0.05% (i.e., 99.95% protein binding). In
other words, if the concentration of tariquidar is 1 nM in the
vascular space, then the free plasma concentration of tariquidar is
0.5 pM. Thus, in a capillary where the local concentration of P-gp
is 40 000 pM, the total bound P-gp in this situation is 4 pM or
0.01%a negligible part of total radioactivity (1 nM).
Based on the above calculations, we hypothesize that the
binding of [11C]tariquidar to P-gp is so effective that it lowers the
concentration of free radioligand in plasma, which then passively
affects the concentration of free radioligand in the brain. One
limitation of this analysis is that it is based on theoretical data and
approximations and thus may not represent the entire in vivo
situation. However, our hypothesis that high, localized density of
P-gp contributes to the low brain signal in PET is supported by
the observation that P-gp knockout mice had slightly higher brain
signal from [11C]tariquidar than wild-type mice did
15
(Figure 2).
This result would be predicted because P-gp knockout mice lack
the receptor that transiently decreases the free concentration of
[11C]tariquidar in capillary plasma; however, parenchymal
uptake is still low because tariquidar is effluxed as a substrate
of BCRP. Taken together, the above calculations and the
observations in mice support a hypothesis, not previously
discussed, that the microenvironment of the capillary affects the
low brain signal obtained from radiolabeled inhibitors in PET
studies of P-gp density.
■CONCLUSIONS
Imaging the density of efflux transporters, such as P-gp, in the
blood−brain barrier using high-affinity inhibitors and PET
presents a particular challenge. Based on our calculations, we
postulate that, while P-gp is highly expressed in brain capillary
endothelial cells, the endothelial cells themselves are present in a
low but relatively uniform density throughout the brain.
Compared with a small region of the brain with locally high
expression of a protein, which can be quantified by PET imaging,
the very low density of P-gp in the brain, high protein binding,
and locally reduced plasma concentrations will require an
imaging agent with a significantly higher affinity for P-gp. More
generally, locally high concentrations of transporters and
receptors in brain endothelial cells do not appear amenable to
imaging due to the vast dilution of endothelial capillary cells in
the brain. The development of radiotracers with picomolar
affinity for targets will be needed to develop diagnostic tools
capable of providing insight into an ever-increasing list of
pathophysiologies associated with dysfunction at the blood−
brain barrier.
■APPENDIX A: CALCULATION OF BMAX (MOL/L) OF
P-GP IN THE WHOLE BRAIN
Values are derived from values found in the literature that can be
generally applied to determine the Bmax of other proteins in brain
endothelial cells. Four values are necessary:
(a) The human brain endothelial cell P-gp expression, which is
6.06 fmol/μg protein (6.06 nmol/g);
28
(b) The protein content of the human brain, which is reported
as ∼10% of total wet weight (i.e., 100 mg protein/g brain),
and fairly uniform across all regions;
40
(c) The density of the human brain, which is 1060 g/L;
41
and
(d) The aforementioned endothelial cell density in the brain of
0.2%.
A concentration of 6.06 nmol P-gp/g of protein (a) is 0.606
nmol P-gp/g brain (10-fold dilution of protein content in cells,
value b), which consequently gives a P-gp concentration of 642
nmol/L (using value c for density of brain). Diluting for
endothelial cells as a component of total brain (0.2%, value d)
gives a final estimated concentration of P-gp in the brain of 1.3
nM.
■APPENDIX B: CALCULATION OF BOUND−FREE
RATIO AND RECEPTOR OCCUPANCY FOR
TARIQUIDAR AND P-GP IN BRAIN CAPILLARIES
Equilibrium Michaelis−Menten:
=
+
B
F
B
KF
max
D
(1)
If F≪KD:
==×
B
F
B
K
Baffinity
max
D
max (2)
where B= concentration of bound radiotracer; F= concentration
of free radiotracer; Bmax = concentration of receptor target; KD=
dissociation constant of radiotracer.
The equilibrium Michaelis−Menten eq 1 is used to determine
the bound (B) concentration of radiotracer given that the other
variables are known. When the free (F) concentration is much
lower than the KDfor the radiotracer (F≪KD), Fcan be
removed (eq 2).
The receptor occupancy (RO), the percentage of receptor
bound by radiotracer, can subsequently be calculated using eq 3:
==
+
B
B
F
KF
RO
max
max D
(3)
Given that in capillaries the Bmax for P-gp and KDfor tariquidar
are known, a range of free tariquidar concentrations (F) can be
used to compute theoretical bound (B) concentration, bound−
free ratio (B/F), and receptor occupancy (RO) of the radiotracer
(see Table A1).
Table A1
Bmax (nM) F(nM) B(nM) B/FRO
40 10 26.67 2.7 66.67%
1 6.67 6.7 16.67%
KD(nM) 0.1 0.78 7.8 1.96%
5 0.01 0.08 8.0 0.20%
0.001 0.01 8.0 0.02%
Molecular Pharmaceutics Review
dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−22292227

■AUTHOR INFORMATION
Corresponding Author
*National Institute of Mental Health, 10 Center Drive, Rm
B1D43, Bethesda, Maryland 20892-1026, United States. Fax: +1
301 480 3610. Tel.: +1 301 594 1368. E-mail: robert.innis@nih.
gov.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This research was supported by the Intramural Research
Programs of the National Institute of Mental Health (Project
Nos. Z01-MH-002852-04 and MH002793-09) and the National
Cancer Institute (Project no. Z01-BC-005598). Oliver Langer’s
studies are funded by the Austrian Science Fund (FWF) project
‘‘Transmembrane Transporters in Health and Disease’’ (SFB
F35) and by the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement number
201380 (“EURIPIDES”). We thank George Leiman for editorial
assistance.
■REFERENCES
(1) Kannan, P.; John, C.; Zoghbi, S. S.; Halldin, C.; Gottesman, M. M.;
Innis, R. B.; Hall, M. D. Imaging the function of P-glycoprotein with
radiotracers: pharmacokinetics and in vivo applications. Clin. Pharm.
Ther. 2009,86 (4), 368−77.
(2) Cooray, H. C.; Blackmore, C. G.; Maskell, L.; Barrand, M. A.
Localisation of breast cancer resistance protein in microvessel
endothelium of human brain. Neuroreport 2002,13 (16), 2059−63.
(3) Graff, C. L.; Pollack, G. M. Drug transport at the blood−brain
barrier and the choroid plexus. Curr. Drug Metab. 2004,5(1), 95−108.
(4) Dombrowski, S. M.; Desai, S. Y.; Marroni, M.; Cucullo, L.;
Goodrich, K.; Bingaman, W.; Mayberg, M. R.; Bengez, L.; Janigro, D.
Overexpression of multiple drug resistance genes in endothelial cells
from patients with refractory epilepsy. Epilepsia 2001,42 (12), 1501−6.
(5) Loscher, W.; Potschka, H. Drug resistance in brain diseases and the
role of drug efflux transporters. Nat. Rev. Neurosci. 2005,6(8), 591−602.
(6) Phelps, M. Molecular Imaging and Its Biological Applications;
Springer: New York, 2004.
(7) Kannan, P.; Brimacombe, K. R.; Kreisl, W. C.; Liow, J. S.; Zoghbi, S.
S.; Telu, S.; Zhang, Y.; Pike, V. W.; Halldin, C.; Gottesman, M. M.; Innis,
R. B.; Hall, M. D. Lysosomal trapping of a radiolabeled substrate of P-
glycoprotein as a mechanism for signal amplification in PET. Proc. Natl.
Acad. Sci. U.S.A. 2011,108 (6), 2593−8.
(8) Piwnica-Worms, D.; Sharma, V. Probing multidrug resistance P-
glycoprotein transporter activity with SPECT radiopharmaceuticals.
Curr. Top. Med. Chem. 2010,10 (17), 1834−45.
(9) Innis, R. B.; Cunningham, V. J.; Delforge, J.; Fujita, M.; Gjedde, A.;
Gunn, R. N.; Holden, J.; Houle, S.; Huang, S. C.; Ichise, M.; Iida, H.; Ito,
H.; Kimura, Y.; Koeppe, R. A.; Knudsen, G. M.; Knuuti, J.; Lammertsma,
A. A.; Laruelle, M.; Logan, J.; Maguire, R. P.; Mintun, M. A.; Morris, E.
D.; Parsey, R.; Price, J. C.; Slifstein, M.; Sossi, V.; Suhara, T.; Votaw, J. R.;
Wong, D. F.; Carson, R. E. Consensus nomenclature for in vivo imaging
of reversibly binding radioligands. J. Cereb. Blood Flow Metab. 2007,27
(9), 1533−9.
(10) Ambudkar, S. V.; Dey, S.; Hrycyna, C. A.; Ramachandra, M.;
Pastan, I.; Gottesman, M. M. Biochemical, cellular, and pharmacological
aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol.
1999,39, 361−98.
(11) Colabufo, N. A.; Berardi, F.; Perrone, M. G.; Capparelli, E.;
Cantore, M.; Inglese, C.; Perrone, R. Substrates, inhibitors and
activators of P-glycoprotein: candidates for radiolabeling and imaging
perspectives. Curr. Top. Med. Chem. 2010,10 (17), 1703−14.
(12) Kannan, P.; Telu, S.; Shukla, S.; Ambudkar, S. V.; Pike, V. W.;
Halldin, C.; Gottesman, M. M.; Innis, R. B.; Hall, M. D. The “Specific”P-
Glycoprotein Inhibitor Tariquidar Is Also a Substrate and an Inhibitor
for Breast Cancer Resistance Protein (BCRP/ABCG2). ACS Chem.
Neurosci. 2011,2(2), 82−9.
(13) Loo, T. W.; Bartlett, M. C.; Detty, M. R.; Clarke, D. M. The
ATPase activity of the P-glycoprotein drug pump is highly activated
when the N-terminal and central regions of the nucleotide-binding
domains are linked closely together. J. Biol. Chem. 2012,287 (32),
26806−16.
(14) Martin, C.; Berridge, G.; Mistry, P.; Higgins, C.; Charlton, P.;
Callaghan, R. The molecular interaction of the high affinity reversal
agent XR9576 with P-glycoprotein. Br. J. Pharmacol. 1999,128 (2),
403−11.
(15) Bauer, F.; Kuntner, C.; Bankstahl, J. P.; Wanek, T.; Bankstahl, M.;
Stanek, J.; Mairinger, S.; Dorner, B.; Loscher, W.; Muller, M.; Erker, T.;
Langer, O. Synthesis and in vivo evaluation of [11C]tariquidar, a
positron emission tomography radiotracer based on a third-generation
P-glycoprotein inhibitor. Bioorg. Med. Chem. 2010,18 (15), 5489−97.
(16) Dorner, B.; Kuntner, C.; Bankstahl, J. P.; Bankstahl, M.; Stanek, J.;
Wanek, T.; Stundner, G.; Mairinger, S.; Loscher, W.; Muller, M.; Langer,
O.; Erker, T. Synthesis and small-animal positron emission tomography
evaluation of [11C]-elacridar as a radiotracer to assess the distribution of
P-glycoprotein at the blood−brain barrier. J. Med. Chem. 2009,52 (19),
6073−82.
(17) Dorner, B.; Kuntner, C.; Bankstahl, J. P.; Wanek, T.; Bankstahl,
M.; Stanek, J.; Mullauer, J.; Bauer, F.; Mairinger, S.; Loscher, W.; Miller,
D. W.; Chiba, P.; Muller, M.; Erker, T.; Langer, O. Radiosynthesis and in
vivo evaluation of 1-[18F]fluoroelacridar as a positron emission
tomography tracer for P-glycoprotein and breast cancer resistance
protein. Bioorg. Med. Chem. 2011,19 (7), 2190−8.
(18) Luurtsema, G.; Schuit, R. C.; Klok, R. P.; Verbeek, J.; Leysen, J. E.;
Lammertsma, A. A.; Windhorst, A. D. Evaluation of [11C]laniquidar as a
tracer of P-glycoprotein: radiosynthesis and biodistribution in rats. Nucl.
Med. Biol. 2009,36 (6), 643−9.
(19) van Waarde, A.; Ramakrishnan, N. K.; Rybczynska, A. A.; Elsinga,
P. H.; Berardi, F.; de Jong, J. R.; Kwizera, C.; Perrone, R.; Cantore, M.;
Sijbesma, J. W.; Dierckx, R. A.; Colabufo, N. A. Synthesis and preclinical
evaluation of novel PET probes for P-glycoprotein function and
expression. J. Med. Chem. 2009,52 (14), 4524−32.
(20) Kawamura, K.; Konno, F.; Yui, J.; Yamasaki, T.; Hatori, A.;
Yanamoto, K.; Wakizaka, H.; Takei, M.; Nengaki, N.; Fukumura, T.;
Zhang, M. R. Synthesis and evaluation of [11C]XR9576 to assess the
function of drug efflux transporters using PET. Ann. Nucl. Med. 2010,24
(5), 403−12.
(21) Kawamura, K.; Yamasaki, T.; Konno, F.; Yui, J.; Hatori, A.;
Yanamoto, K.; Wakizaka, H.; Ogawa, M.; Yoshida, Y.; Nengaki, N.;
Fukumura, T.; Zhang, M. R. Synthesis and in vivo evaluation of (1)(8)F-
fluoroethyl GF120918 and XR9576 as positron emission tomography
probes for assessing the function of drug efflux transporters. Bioorg. Med.
Chem. 2011,19 (2), 861−70.
(22) Kawamura, K.; Yamasaki, T.; Konno, F.; Yui, J.; Hatori, A.;
Yanamoto, K.; Wakizaka, H.; Takei, M.; Kimura, Y.; Fukumura, T.;
Zhang, M. R. Evaluation of limiting brain penetration related to P-
glycoprotein and breast cancer resistance protein using [(11)C]-
GF120918 by PET in mice. Mol. Imaging Biol. 2011,13 (1), 152−60.
(23) Mairinger, S.; Langer, O.; Kuntner, C.; Wanek, T.; Bankstahl, J. P.;
Bankstahl, M.; Stanek, J.; Dorner, B.; Bauer, F.; Baumgartner, C.;
Loscher, W.; Erker, T.; Muller, M. Synthesis and in vivo evaluation of the
putative breast cancer resistance protein inhibitor [11C]methyl 4-((4-
(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl)phenyl)-
amino-car Bonyl)-2-(quinoline-2-carbonylamino)benzoate. Nucl. Med.
Biol. 2010,37 (5), 637−44.
(24) Mairinger, S.; Wanek, T.; Kuntner, C.; Doenmez, Y.; Strommer,
S.; Stanek, J.; Capparelli, E.; Chiba, P.; Muller, M.; Colabufo, N. A.;
Langer, O. Synthesis and preclinical evaluation of the radiolabeled P-
glycoprotein inhibitor [(11)C]MC113. Nucl. Med. Biol. 2012,39 (8),
1219−25.
(25) Halldin, C.; Gulyas, B.; Langer, O.; Farde, L. Brain radioligands
state of the art and new trends. Q. J. Nucl. Med. 2001,45 (2), 139−52.
(26) Eckelman, W. C.; Mathis, C. A. Targeting proteins in vivo: in vitro
guidelines. Nucl. Med. Biol. 2006,33 (2), 161−4.
Molecular Pharmaceutics Review
dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−22292228

(27) Luurtsema, G.; Verbeek, G. L.; Lubberink, M.; Lammertsma, A.
A.; Dierckx, R.; Elsinga, P.; Windhorst, A. D.; van Waarde, A. Carbon-11
labeled tracers for in vivo imaging P-glycoprotein function: kinetics,
advantages and disadvantages. Curr. Top. Med. Chem. 2010,10 (17),
1820−33.
(28) Kamiie, J.; Ohtsuki, S.; Iwase, R.; Ohmine, K.; Katsukura, Y.;
Yanai, K.; Sekine, Y.; Uchida, Y.; Ito, S.; Terasaki, T. Quantitative atlas of
membrane transporter proteins: development and application of a
highly sensitive simultaneous LC/MS/MS method combined with
novel in-silico peptide selection criteria. Pharm. Res. 2008,25 (6),
1469−83.
(29) Ito, K.; Uchida, Y.; Ohtsuki, S.; Aizawa, S.; Kawakami, H.;
Katsukura, Y.; Kamiie, J.; Terasaki, T. Quantitative membrane protein
expression at the blood−brain barrier of adult and younger cynomolgus
monkeys. J. Pharm. Sci. 2011,100 (9), 3939−50.
(30) Uchida, Y.; Ohtsuki, S.; Katsukura, Y.; Ikeda, C.; Suzuki, T.;
Kamiie, J.; Terasaki, T. Quantitative targeted absolute proteomics of
human blood−brain barrier transporters and receptors. J. Neurochem.
2011,117 (2), 333−45.
(31) Larsson, H. B.; Courivaud, F.; Rostrup, E.; Hansen, A. E.
Measurement of brain perfusion, blood volume, and blood−brain
barrier permeability, using dynamic contrast-enhanced T(1)-weighted
MRI at 3 T. Magn. Reson. Med. 2009,62 (5), 1270−81.
(32) Phelps, M. E.; Huang, S. C.; Hoffman, E. J.; Kuhl, D. E. Validation
of tomographic measurement of cerebral blood volume with C-11-
labeled carboxyhemoglobin. J. Nucl. Med. 1979,20 (4), 328−34.
(33) Wiederhold, K. H.; Bielser, W., Jr.; Schulz, U.; Veteau, M. J.;
Hunziker, O. Three-dimensional reconstruction of brain capillaries from
frozen serial sections. Microvasc. Res. 1976,11 (2), 175−80.
(34) Pardridge, W. M. Blood-brain barrier biology and methodology. J.
Neurovirol. 1999,5(6), 556−69.
(35) Pardridge, W. Peptide Drug Delivery to the Brain; Raven Press:
New York, 1991; p 357.
(36) Hicks, P.; Rolsten, C.; Brizzee, D.; Samorajski, T. Age-related
changes in rat brain capillaries. Neurobiol. Aging 1983,4(1), 69−75.
(37) Pardridge, W. M.; Boado, R. J.; Farrell, C. R. Brain-type glucose
transporter (GLUT-1) is selectively localized to the blood−brain
barrier. Studies with quantitative western blotting and in situ
hybridization. J. Biol. Chem. 1990,265 (29), 18035−40.
(38) Mairinger, S.; Erker, T.; Muller, M.; Langer, O. PET and SPECT
radiotracers to assess function and expression of ABC transporters in
vivo. Curr. Drug Metab. 2011,12 (8), 774−92.
(39) Zoghbi, S. S.; Anderson, K. B.; Jenko, K. J.; Luckenbaugh, D. A.;
Innis, R. B.; Pike, V. W. On quantitative relationships between drug-like
compound lipophilicity and plasma free fraction in monkey and human.
J. Pharmaceut. Sci. 2012,101 (3), 1028−39.
(40) Banay-Schwartz, M.; Kenessey, A.; DeGuzman, T.; Lajtha, A.;
Palkovits, M. Protein content of various regions of rat brain and adult
and aging human brain. Age 1992,15 (2), 51−54.
(41) Witelson, S. F.; Beresh, H.; Kigar, D. L. Intelligence and brain size
in 100 postmortem brains: sex, lateralization and age factors. Brain 2006,
129 (Pt 2), 386−98.
(42) Melchior, D. L.; Sharom, F. J.; Evers, R.; Wright, G. E.; Chu, J. W.;
Wright, S. E.; Chu, X.; Yabut, J. Determining P-glycoprotein-drug
interactions: evaluation of reconstituted P-glycoprotein in a liposomal
system and LLC-MDR1 polarized cell monolayers. J. Pharmacol. Toxicol.
Meth. 2012,65 (2), 64−74.
(43) Ferry, D. R.; Russell, M. A.; Kerr, D. J.; Correa, I. D.; Prakash, S. R.
[3H]-GG918 (GF120918) binds with positive co-operativity to human
P-glycoprotein with a nM dissociation constant. Proc. Am. Assoc. Cancer
Res. 1996,37, 2246.
Molecular Pharmaceutics Review
dx.doi.org/10.1021/mp400011g |Mol. Pharmaceutics 2013, 10, 2222−22292229