Synthesis and in vivo evaluation of [11C]tariquidar, a positron emission tomography radiotracer based on a third-generation P-glycoprotein inhibitor

Article abstract of DOI:10.1016/j.bmc.2010.06.057

The aim of this study was to develop a positron emission tomography (PET) tracer based on the dual P-glycoprotein (P-gp) breast cancer resistance protein (BCRP) inhibitor tariquidar (1) to study the interaction of 1 with P-gp and BCRP in the blood-brain b

Full text of DOI:10.1016/j.bmc.2010.06.057

Bioorganic & Medicinal Chemistry 18 (2010) 5489–5497
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vivo evaluation of [11C]tariquidar, a positron emission
tomography radiotracer based on a third-generation P-glycoprotein inhibitor
Florian Bauer b,c, , Claudia Kuntner a, , Jens P. Bankstahl d, Thomas Wanek a, Marion Bankstahl d,
Johann Stanek a,c, Severin Mairinger a,b,c, Bernd Dörner b, Wolfgang Löscher d, Markus Müller c,
a,c
Thomas Erker b, , Oliver Langer
*
a Molecular Medicine, AIT Austrian Institute of Technology GmbH, Seibersdorf, Austria
b Department of Medicinal Chemistry, University of Vienna, Austria
c Department of Clinical Pharmacology, Medical University of Vienna, Austria
d Department of Pharmacology, Toxicology & Pharmacy, University of Veterinary Medicine Hanover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this study was to develop a positron emission tomography (PET) tracer based on the dual P-gly-
coprotein (P-gp) breast cancer resistance protein (BCRP) inhibitor tariquidar (1) to study the interaction of 1
with P-gp and BCRP in the blood–brain barrier (BBB) in vivo. O-Desmethyl-1 was synthesized and reacted
with [11C]methyl triflate to afford [11C]-1. Small-animal PET imaging of [11C]-1 was performed in naïve rats,
before and after administration of unlabeled 1 (15 mg/kg, n = 3) or the dual P-gp/BCRP inhibitor elacridar
(5 mg/kg, n = 2), as well as in wild-type, Mdr1a/b(À/À), Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À) mice (n = 3).
In vitro autoradiography was performed with [11C]-1 using brain sections of all four mouse types, with
Received 12 April 2010
Revised 10 June 2010
Accepted 16 June 2010
Available online 22 June 2010
Keywords:
PET
and without co-incubation with unlabeled 1 or elacridar (1 lM). In PET experiments in rats, administration
[
11C]tariquidar
of unlabeled 1 or elacridar increased brain activity uptake by a factor of 3–4, whereas blood activity levels
remained unchanged. In Mdr1a/b(À/À), Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À) mice, brain-to-blood ratios of
activity at 25 min after tracer injection were 3.4, 1.8 and 14.5 times higher, respectively, as compared to
wild-type animals. Autoradiography showed approximately 50% less [11C]-1 binding in transporter knock-
out mice compared to wild-type mice and significant displacement by unlabeled elacridar in wild-type and
Mdr1a/b(À/À) mouse brains. Our data suggest that [11C]-1 interacts specifically with P-gp and BCRP in the
BBB. However, further investigations are needed to assess if [11C]-1 behaves in vivo as a transported or a
non-transported inhibitor.
P-glycoprotein
Breast cancer resistance protein
Blood-brain barrier
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
potentially harmful lipophilic compounds from natural sources
and other lipophilic xenobiotics that otherwise could penetrate
The ATP-binding cassette (ABC) transporter P-glycoprotein
(P-gp, ABCB1) was initially discovered in the 1970s as an active ef-
flux transporter involved in the multidrug resistance of cancer
cells, and was the first drug efflux transporter to be detected in
endothelial cells at the human blood–brain barrier (BBB).1,2 It has
been suggested that P-gp serves as a general defense mechanism
in the mammalian BBB, protecting the brain from intoxication by
the BBB by simple diffusion without any limitation.3 Regional over-
activity of P-gp and related transporters in the BBB contributes to
the phenomenon of drug resistance in neurological disorders, such
as epilepsy and depression, by impeding therapeutically effective
concentrations of central nervous system (CNS) drugs at their sites
of action.3
A promising strategy to enhance drug penetration across the
BBB and thereby overcome drug resistance is the administration
of third-generation P-gp inhibitors, which were originally devel-
oped as an adjunctive therapy for drug-resistant cancers and are
characterized by a high potency in the nanomolar range, lack of
toxicity and lack of cytochrome P450 interactions.4 The anthranilic
acid derivative tariquidar (1, Scheme 1) is one of the most potent
and selective third-generation P-gp inhibitors known to date.5
Unlike first- and second-generation P-gp inhibitors, such as verap-
amil, cyclosporine A or valspodar (PSC 833), 1 is a non-competitive
Abbreviations: BBB, blood–brain barrier; BCRP, breast cancer resistance protein;
Bcrp1(À/À) mouse, BCRP knockout mouse; Mdr1a/b(À/À) mouse, P-gp knockout
mouse; Mdr1a/b(À/À)Bcrp1(À/À) mouse, P-gp/BCRP knockout mouse; PET, positron
emission tomography; P-gp, P-glycoprotein; SPE, solid-phase extraction; SUV,
standardized uptake value; TAC, time-activity curve.
* Corresponding author. Tel.: +43 1 4277 550 03; fax: +43 1 4277 9 551.
E-mail addresses: thomas.erker@univie.ac.at (T. Erker), oliver.langer@ait.ac.at
(O. Langer).
Florian Bauer and Claudia Kuntner contributed equally to this study.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.057

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F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
O
O
O
O
OH
O
O
O
A
B
O
N
HO
HO
O2N
O
O
O
O
O2N
O
O
N
H
4
2
3
N
O2N
NH2
O
O
O
O
C
D
O
O
N
N
O
O
O
O
O
N
N
H
H
H2N
HN
O
5
7
6
O
N
O
O
O
E
F
N
N
O
O
O
O
11CH3
OH
O
N
N
H
H
HN
HN
O
[11C]-1
O
O
N
N
Scheme 1. Synthesis of precursor 7 and its radiolabeling with [11C]methyl triflate to give [11C]-1. Reagents: (A) pivalic anhydride, DMF, Cs2CO3; (B) SOCl2, THF, Et3N; (C) H2,
Pd/C, EtOAc/MeOH; (D) 3-quinolinecarbonyl chloride, THF, Et3N; (E) NH3, EtOH, 50 °C; (F) [11C]methyl triflate, aq NaOH, acetone, 60 °C.
inhibitor and not a substrate of P-gp.6 Compound 1 also inhibits
breast cancer resistance protein (BCRP, ABCG2), another ABC-type
transporter expressed in the BBB, but at higher concentrations
than those at which it inhibits P-gp.7 However, unlike first-and
second-generation P-gp inhibitors, 1 does not inhibit multidrug
resistance-associated proteins (MRPs), such as MRP1.8 Compound
1 was initially tested in clinical trials in cancer patients in
combination with anticancer drugs that are P-gp substrates (e.g.,
paclitaxel, docetaxel, doxorubicin, vinorelbine), which demon-
strated treatment response in some patients.5 However, some of
these trials had to be discontinued because of increased toxicity
of treatment regimens, which has been attributed to enhanced
systemic exposure to anticancer drugs due to peripheral P-gp
inhibition.4,5
(R)-[11C]verapamil was measured by PET imaging in healthy sub-
jects.14 Our study showed a modest but significant (+24 15%) in-
crease in (R)-[11C]verapamil brain distribution volume, which
suggested that higher doses of 1 might be needed in the clinic in
order to improve the clinical efficacy of CNS drugs in therapy-
refractory patients. On the other hand, in rats comparable plasma
levels of 1 to those achieved in humans in our study caused an in-
crease of approximately +300% in (R)-[11C]verapamil brain distri-
bution volume, which points to pronounced species difference in
tariquidar-induced P-gp inhibition.15 Another human study as-
sessed the effect of different doses of 1 on brain penetration of
the P-gp substrate radiotracer [11C]-N-desmethyl-loperamide and
found a fourfold increase in brain radioactivity uptake after
6 mg/kg of 1.16
In contrast to its well-studied role in blocking P-gp in chemore-
sistant cancer cells, considerably less is known about the capacity
of 1 to inhibit P-gp in the BBB and thereby facilitate brain entry of
CNS drugs. A number of animal studies have shown that brain lev-
els of antiepileptics, cytostatics and other CNS drugs were in-
creased following administration of 1.9–12 Only few human
studies assessing the effect of 1 on P-gp in the BBB have been con-
ducted to date. Kurnik et al. studied brain penetration of the P-gp
substrate loperamide by measuring reduction in pupil diameter as
a surrogate marker for central opioid effect, with and without co-
administration of 1 at a dose of 2 mg/kg body weight.13 This study
failed to demonstrate an effect of 1 on loperamide’s central effects,
whereas inhibition of P-gp in lymphocytes was >90%, suggesting
that P-gp in the BBB is more resistant to inhibition as compared
to peripheral P-gp.10 In a study recently conducted at our center,
the effect of 1 (2 mg/kg) on brain penetration of the P-gp substrate
An alternative approach to using radiolabeled P-gp substrates,
such as (R)-[11C]verapamil,12,14 11C]-N-desmethyl-loperamide,17,18 or
[
6,7-dimethoxy-2-[3-(5-[11C]methoxy-1,2,3,4-tetrahydronaphthalen-1-
yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline ([11C]MC-266)19 for study-
ing the interaction of 1 with P-gp at the BBB would be to perform
PET experiments directly with radiolabeled 1. Such a radiotracer
might, for example, help to better understand species differences in
tariquidar-induced P-gp inhibition as well as the differential sensitiv-
ity of cerebral and peripheral P-gp to inhibition. As opposed to P-gp
substrate radiotracers, radiolabeled 1 is expected to bind to P-gp at
the BBB without being transported by it and thereby allow mapping
of the distribution of cerebral P-gp.
The aim of this study was to develop carbon-11-labeled 1
([11C]-1) as a PET tracer to directly study the interaction of 1 with
P-gp and BCRP in vivo. Here, we report on the precursor synthesis,
11C-labeling and small-animal PET evaluation of [11C]-1.

F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
5491
2. Results
2.1. Chemistry and radiolabeling
The radiolabeling precursor of [11C]-1, O-desmethyl-tariquidar
(7), was synthesized as outlined in Scheme 1. Commercially avail-
able 4,5-dimethoxy-2-nitrobenzoic acid was first mono-demethy-
lated using aq KOH to yield the corresponding 5-hydroxy
derivative 2 (see Supplementary data).20 Derivative 2 was then
pivaloyl-protected to give compound 3 (see Supplementary data),
which was transformed into its acid chloride and coupled with
6,7-dimethoxy-2-(4-aminophenethyl)-1,2,3,4-tetrahydroisochino-
line to afford compound 4.21,22 The NO2 group in 4 was reduced by
catalytic hydrogenation to obtain the corresponding anthranilic
acid derivative 5, which was reacted with 3-quinolinecarbonyl
chloride to afford O-pivaloyl-tariquidar (6). The pivaloyl protective
group in 6 was removed by NH3 in EtOH to give 7. For 11C-labeling,
7 was reacted with [11C]methyl triflate in acetone containing aq
NaOH. [11C]-1 ready for iv injection was obtained in a decay-cor-
rected radiochemical yield of 5.4 3.0% (n = 45), based on
[
11C]CH4, in a total synthesis time of approximately 40 min. Radio-
chemical purity was greater than 98% (see Supplementary data)
and specific activity at end of synthesis was 114 67 GBq/lmol
(n = 13). The identity of [11C]-1 was confirmed by HPLC co-injec-
tion with unlabeled 1 (see Supplementary data).
2.2. Small-animal PET in rats and mice
[
11C]-1 was evaluated in rats by performing paired PET scans:
a baseline scan (scan 1) and a second scan after administration of
unlabeled 1 or elacridar (scan 2) (Fig. 1). In scan 1, brain uptake
of activity was low. Peak brain uptake was 0.71 0.14 (standard-
ized uptake value, SUV) at 0.5–1.3 min after tracer injection.
At 25 min after tracer injection, brain activity uptake had declined
to 0.19 0.02. In response to administration of unlabeled 1
(15 mg/kg) or elacridar (5 mg/kg) at 60 min after injection of
Figure 1. TACs (mean SUV standard deviation, SD) in whole brain (scan 1: open
squares, scan 2: filled squares) and arterial blood (scan 1: open triangles, scan 2:
filled triangles) for paired PET scans in rats using (A) unlabeled 1 (15 mg/kg, n = 3)
or (B) elacridar (5 mg/kg, n = 2) as inhibitor. Inhibitor was administered as an iv
bolus at 60 min after start of scan 1 (see timeline above).
[
11C]-1 there was a steep increase in brain activity concentration,
reaching SUVs of 0.43 0.03 and 0.71 0.06, respectively, at
130 min after tracer injection, whereas blood activity levels
appeared unchanged (Fig. 1). In scan 2, performed after injection
of unlabeled 1 or elacridar, brain activity uptake at 25 min after
tracer injection was 2.9 0.8 and 4.3 1.2 times higher as com-
pared to scan 1, respectively, with similar blood activity levels
as in scan 1 (Fig. 1). In Figure 2, representative PET summation
images are shown for scans recorded before and after inhibitor
administration.
activity rose constantly throughout the scan duration. Brain activ-
ity uptake at 25 min after tracer injection was normalized to mean
blood activity levels measured in separate groups of animals.
Brain-to-blood ratios of activity at 25 min after tracer injection
were 3.4 (p = 0.0006), 1.8 (p = 0.0009) and 14.5 times (p = 0.0002)
higher in Mdr1a/b(À/À), Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À) mice,
respectively, as compared to wild-type animals (brain-to-blood
Radiometabolites of [11C]-1 in plasma were assessed by a solid-
phase extraction (SPE) assay. At 20 min after [11C]-1 injection,
4
4% of total plasma activity was recovered in SPE fractions 1 and
2 (corresponding to polar radiometabolites of [11C]-1) and 96 4%
in fraction 3 (n = 8, mean values of 20-min samples collected in scans
1 and 2). At 60 min after second [11C]-1 injection, 4 5% of total plas-
ma activity was in fractions 1 and 2, and 96 5% in fraction 3. Frac-
tion 3 was further analyzed by radio-high-performance liquid
chromatography (HPLC), which suggested that unchanged [11C]-1
was the only radioactive species present, even though it cannot be
excluded that radiometabolites of [11C]-1 were hidden under the
broad HPLC peak of [11C]-1. Representative HPLC chromatograms
for samples collected at 20 and 60 min after tracer injection are
shown in the Supplementary data.
ratios, wild-type: 1.0 0.1; Mdr1a/b(À/À)
: :
3.3 0.4; Bcrp1(À/À)
1.8 0.1; Mdr1a/b(À/À)Bcrp1(À/À): 14.4 1.7).
2.3. In vitro autoradiography
Figure 4 shows results from in vitro autoradiography of brain
sections from all four mouse types, performed with [11C]-1 alone
or with mixtures of [11C]-1 and unlabeled 1 or elacridar. Compared
to wild-type mouse brain, mean binding of [11C]-1 was 49%
(p = 0.003), 41% (p = 0.006) and 46% (p = 0.001) lower in Mdr1a/
In Figure 3, PET summation images and brain time-activity
curves (TACs) of [11C]-1 in wild-type, Mdr1a/b(À/À), Bcrp1(À/À) and
Mdr1a/b(À/À)Bcrp1(À/À) mice are shown. Brain TACs were higher in
all transporter knockout mice as compared to wild-type animals
(Fig. 3B). In Mdr1a/b(À/À)Bcrp1(À/À) mice, brain TACs were strikingly
different as compared to the other three mouse types in that brain
b
(À/À), Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À) mouse brain, respec-
tively (Fig. 4A and B). Co-incubation of [11C]-1 with unlabeled 1
significantly reduced binding of [11C]-1 to brain sections of all four
mouse types (Fig. 4C). Co-incubation with elacridar, on the other
hand, only caused significant reductions of [11C]-1 binding in
wild-type and Mdr1a/b(À/À) mouse brain (Fig. 4C).

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F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
Figure 2. Exemplary coronal (left), horizontal (center) and sagittal (right) PET summation images in rats, (A) before administration of unlabeled inhibitor (scan 1: 0–60 min),
(B) after administration of unlabeled 1 (15 mg/kg,), and (C) after administration of elacridar (5 mg/kg). In (A) and (B) the same rat is shown. Activity concentration is
expressed as SUV, and the radiation scale is set from 0.05 to 2.5. Anatomical structures are indicated by arrows (br, brain; e, eye; sg, submandibular gland; pg, pituitary gland;
hg, haderian gland).
A
Wild-type
Mdr1a/b(-/-)
Bcrp1(-/-)
Mdr1a/b(-/-)Bcrp1(-/-)
1.6
B
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-10
0
10
20
30
40
50
60
Time (min)
Figure 3. (A) Exemplary sagittal PET summation images (0–60 min) of wild-type, Mdr1a/b(À/À), Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À) mice. Activity concentration is expressed
as SUV, and the radiation scale is set from 0.05 to 2.5. Anatomical structures are indicated by arrows (br, brain; sg, submandibular gland). (B) TACs (mean SUV SD, n = 3 per
mouse type) in whole brain of wild-type (open squares), Mdr1a/b(À/À) (filled squares), Bcrp1(À/À) (filled triangles) and Mdr1a/b(À/À)Bcrp1(À/À) mice (filled circles).

F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
5493
Mdr1a/b(-/-)
Bcrp1(-/-)
wild-type
Mdr1a/b(-/-)
Bcrp1(-/-)
A
max
min
9.E+06
B
8.E+06
7.E+06
6.E+06
5.E+06
4.E+06
3.E+06
2.E+06
1.E+06
0.E+00
wild-type Mdr1a/b(-/-) Bcrp1(-/-) Mdr1a/b(-/-)
Bcrp1(-/-)
ns
C
ns
**
140%
120%
100%
80%
60%
40%
20%
0%
*
***
***
***
***
wild-type
Mdr1a/b(-/-)
Bcrp1(-/-)
Mdr1a/b(-/-)
Bcrp1(-/-)
Figure 4. (A) In vitro autoradiograms of coronal brain slices (10
row), with [11C]-1 and unlabeled 1 (1 M, second row) and with [11C]-1 and unlabeled elacridar (1
luminiscence units SD) determined by in vitro autoradiography. (C) Displacement of [11C]-1 binding to mouse brain sections (mean% SD) (white bars) by unlabeled 1
l
m) of wild-type, Mdr1a/b(À/À), Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À) mice incubated with [11C]-1 alone (first
M, third row). (B) Binding of [11C]-1 to mouse brain sections (mean digital
l
l
*
**
***
(hatched bars) or elacridar (black bars) ( , p <0.05; , p <0.005; , p <0.0005; ns, not significant; 2-tailed t-test).
different approaches. First, we performed PET experiments in naïve
3. Discussion
rats before and after administration of unlabeled 1 or the potent
dual P-gp/BCRP inhibitor elacridar. Second, we performed PET as
well as in vitro autoradiography in wild-type mice and in Mdr1a/
The aim of this study was to label 1 with a positron-emitting
radionuclide and to assess the interaction of radiolabeled 1 with
P-gp and BCRP in the BBB. As 1 contains four OCH3 groups, the
most straightforward radiolabeling approach was O-11C-methyla-
tion of O-desmethyl-1, which offers the advantage that the radio-
tracer retains the chemical structure of native 1 (Scheme 1).
During the preparation of this manuscript a study by Kawamura
et al. has appeared in the literature, which also reported on the
11C-labeling of 1, but in a different labeling position than in our
work (i.e., on the dimethoxy-tetrahydroisoquinolinylethyl part of
the molecule).23 The in vivo specificity of [11C]-1 for P-gp and BCRP
in the BBB was assessed by small-animal PET imaging using two
b
(À/À), Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À) mice, which do not ex-
press the pharmacological targets of [11C]-1.
3.1. Is [11C]-1 transported by P-gp?
Based on in vitro experiments with 1, Mistry et al.8 first suggested
that 1 is not transported by P-gp, which was substantiated by subse-
quentin vitrostudies,6,24 indicatingthat 1 is a non-transported mod-
ulator of P-gp, which inhibits P-gp functionby binding at a site that is
distinct from the site(s) of interaction of transported substrates.6,25

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F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
However, the in vivo behavior of [11C]-1 was comparable to what we
had previously seen for the P-gp substrate (R)-[11C]verapamil.12,15 In
immediate response to injection of unlabeled 1 or elacridar, which
was administered during [11C]-1 baseline PET scans in rats, brain
activity started to rise significantly, with greater increases seen after
administration of elacridar (Fig. 1B) than after 1 (Fig. 1A). In the sec-
ond PET scan, performed after administration of unlabeled inhibitor,
brain activity uptake was 3–4 times greater as compared to baseline
scans (Figs. 1 and 2). Interestingly, similar results were obtained
with [11C]elacridar, another radiolabeled P-gp inhibitor developed
in our laboratory.26 Moreover, the P-gp inhibitor [11C]laniquidar
showed 6–9 times higher brain activity uptake after pretreatment
with cyclosporine A (50 mg/kg, iv) in biodistribution studies in
rats.27 Also in transgenic mice, which lacked P-gp and/or BCRP, brain
uptake of activity following injection of [11C]-1 was greater than in
wild-type animals, which expressed both transporters in the BBB
(Fig. 3).
[
11C]-1 binding by elacridar was seen, but it was clearly less pro-
nounced than in wild-type mice. Unlabeled 1, on the other hand,
displaced [11C]-1 binding from all four types of mouse brain sections,
apparently independent of the presence or absence of P-gp and/or
BCRP. The reasons for this lack of differentiation of [11C]-1 displace-
ment by unlabeled 1 in the four studied mouse types remain unclear
at present but might suggest that [11C]-1 additionally binds to an-
other saturable binding site in mouse brain tissue that is different
from P-gp and BCRP. However, this site will probably be irrelevant
for PET imaging due to the gatekeeper function of P-gp/BCRP. Our
assumption that [11C]-1 binds to P-gp and is not transported by
P-gp is further supported by pilot data acquired in a rat model of
P-gp overexpression,30 which revealed increased brain uptake of
[
11C]-1 as compared to naïve rats, whereas brain uptake of the
P-gp substrate (R)-[11C]verapamil was decreased.31
3.2. Does [11C]-1 selectively interact with P-gp in the BBB?
Taken together, these observations may suggest that [11C]-1 be-
haves in vivo as a P-gp substrate rather than as a non-transported
P-gp inhibitor. On the other hand, several lines of evidence suggest
that 1 is a non-transported inhibitor of P-gp. According to Polli
et al. the criteria for classifying a compound as a non-transported
P-gp inhibitor are (i) a basolateral-apical (B-A) to apical-basolateral
(A-B) concentration ratio <2 in monolayer transport assays, (ii) lack
of adenosine triphosphatase activity stimulation and (iii) inhibition
of substrate transport.28 Compound 1 seems to meet all three cri-
teria,6,24,25 but additional experiments with 3H-labeled 1 might be
warranted in the future to fully resolve this issue.
PET experiments in wild-type and Mdr1a/b(À/À), Bcrp1(À/À) and
Mdr1a/b(À/À)Bcrp1(À/À) mice suggested that the contribution of
P-gp to limiting brain entry of [11C]-1 was greater than the contri-
bution of BCRP, as reflected by approximately two times higher
brain-to-blood ratios of activity in Mdr1a/b(À/À) compared to
Bcrp1(À/À) mice (Fig. 3). On the other hand, the combined effect
of P-gp and BCRP knockout (Mdr1a/b(À/À)Bcrp1(À/À) mice) on brain
uptake of [11C]-1 was far greater than the sum of its individual con-
tributions in Mdr1a/b(À/À) and Bcrp1(À/À) mice. This observation has
also been made by other investigators for other P-gp/BCRP sub-
strate drugs23,32,33 and points to a concerted action of P-gp and
BCRP in the BBB. In the absence of P-gp, BCRP may take over its role
in limiting brain entry of dual substrates and vice versa. Moreover,
it cannot be ruled out that Mdr1a/b(À/À) mice, as a compensatory
mechanism, show increased levels of BCRP in the BBB34 and vice
versa. Data from in vitro autoradiography suggested that [11C]-1
binds to both P-gp and BCRP, but were not able to discern the indi-
vidual contributions of the two transporters to [11C]-1 binding.
A possible explanation for the in vivo results obtained with [11C]-
1 might be that a tracer dose of [11C]-1, which contains <1
lg of unla-
beled 1, binds with nanomolar affinity (equilibrium dissociation
constantKD: 5.1 nM6) and a slow rate ofdissociation toP-gp and pos-
sibly BCRP, which both act as gatekeepers at the BBB and thereby
limit diffusion of [11C]-1 into brain.26 Administration of unlabeled
1 or elacridar might have displaced [11C]-1 from its P-gp/BCRP bind-
ing site, resulting in unhindered passive diffusion of unmetabolized
[
11C]-1 from blood into brain. As brain activity levels rose above
blood levels during the later part of scan 1 (Fig. 1), it can be assumed
that diffusion into brain was driven by one or more so far unknown
binding processes in brain, which were apparently not saturated at
the administered doses of unlabeled 1 or elacridar. Displacement
of P-gp binding of 3H-labeled 1 by elacridar has been reported be-
fore.6,29 As elacridar and 1 appear to have distinct binding sites on
P-gp, displacement of [11C]-1 binding by elacridar might have been
caused by allosteric modulation.29 Interestingly, we had previously
seen only a weak increase in brain activity uptake during PET scans
with [11C]elacridar when unlabeled 1 was administered,26 but this
might be related to the fact that the employed dose of 1 (3 mg/kg)
did not completely inhibit P-gp.15
3.3. Is [11C]-1 useful as a PET tracer for mapping the distribution
of P-gp/BCRP at the BBB?
P-gp is a target protein which is ubiquitously and abundantly
expressed in brain tissue. Kamiie et al. report a density (Bmax) of
the Mdr1a peptide of 15 fmol/lg protein in mouse brain capillaries,
which translates to a value of 75 nM when assuming a vascular vol-
ume in brain tissue of approximately 5% and a protein content of
brain capillaries of approximately 10%.35 The ratio of the Bmax of a
given target protein to the KD of a prospective radioligand is
commonly used as a predictor of the target-to-nontarget ratio to
be expected for in vivo imaging.36 Based on a KD value for binding
of trititated 1 to Chinese hamster ovary resistant cells (CHrB30) of
5.1 nM,6 the Bmax/KD is approximately 15 for 1, which should allow
for obtaining a sufficiently high in vivo signal with [11C]-1. Despite
this Bmax/KD ratio, the PET signal obtained with [11C]-1 was low
(brain uptake (SUV) 0.19 0.02 at 25 min after tracer injection into
rats), which might not be optimal for in vivo imaging and limit an
accurate quantification of P-gp/BCRP expression levels with [11C]-1.
We hypothesize that the low brain uptake of [11C]-1 might re-
sult from the gatekeeper function of P-gp/BCRP, which prevents
brain entry of the radiolabeled probe, presumably by P-gp/BCRP-
specific binding. A possible advantage of the gatekeeper function
of P-gp/BCRP might be that it prevents non-specific binding of
the highly lipophilic probe (log P 5.52) in brain tissue, which would
mask P-gp/BCRP-specific binding. Looking at the brain TACs in
Mdr1a/b(À/À)Bcrp1(À/À) mice (Fig. 3) gives a good impression of
the high level of non-specific binding that would be obtained with
In order to further substantiate our assumption of P-gp/BCRP-
specific in vivo binding of [11C]-1, in vitro autoradiography of mouse
brain sections was performed with [11C]-1. Importantly, brain sec-
tions of knockout mice, which lacked either P-gp or BCRP or both,
showed approximately 50% less binding of [11C]-1 as compared to
wild-type mouse brain (Fig. 4A and B). This is exactly the opposite
of what we had seen in the in vivo experiments(Fig. 3), where knock-
out mice showed higher brain activity uptake than wild-type mice.
As the BBB does not act as gatekeeper in the autoradiography set-
up lower binding of [11C]-1 in knockout mice most likely reflects
the absence of P-gp and/or BCRP. Furthermore, the specificity of
[
11C]-1 binding to mouse brain sectionswas investigated by co-incu-
bation with unlabeled 1 or elacridar. The dual P-gp/BCRP inhibitor
elacridar was able to significantly displace [11C]-1 binding in mouse
brain sections expressing P-gp and BCRP (wild-type mice), but not in
brain sections of Mdr1a/b(À/À)Bcrp1(À/À) and Bcrp1(À/À) mice (Fig. 4C).
In Mdr1a/b(À/À) mouse brain sections, significant displacement of
[
11C]1 if it was not for the gatekeeper function of P-gp/BCRP.

F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
5495
If the small brain PET signal obtained with [11C]-1 is indeed spe-
cific to P-gp/BCRP in the BBB, the apparent lack of non-specific
binding might therefore still make [11C]-1 useful for imaging trans-
porter distribution at the BBB. Along the same lines, the apparently
good metabolic stability of [11C]-1 (96% unchanged parent in plas-
ma at 60 min after injection of [11C]-1 to rats) can be considered as
an advantage, as radiometabolites of [11C]-1, if present, might
show P-gp/BCRP-independent brain uptake, such as is the case
for (R)-[11C]verapamil,37 and thereby potentially contaminate the
P-gp/BCRP-specific signal of [11C]-1. The metabolic stability of
Welfare Committee and all study procedures were performed in
accordance with the Austrian Animal Experiments Act.
4.3. 5-(4-[2-(6,7-Dimethoxy-1,2,3,4-tetrahydro-2-isoquin
olinyl)ethyl]anilinocarbonyl)-2-methoxy-4-nitrophenyl
pivalate (4)
5-[(2,2-Dimethylpropanoyl)oxy]-4-methoxy-2-nitrobenzoic
acid (3, 0.68 g, 2.29 mmol), which had been prepared from 5-hy-
droxy-4-methoxy-2-nitrobenzoic acid (2) as described in the Sup-
plementary data, was refluxed in SOCl2 (4 mL) for 3 h. Excess SOCl2
was evaporated. The acid chloride of 3 was dissolved in THF and
slowly added to a solution of 6,7-dimethoxy-2-(4-aminopheneth-
yl)-1,2,3,4-tetrahydroisochinoline (0.68 g, 2.17 mmol) in THF con-
taining Et3N (1 mL), which had been prepared as described
previously.26 The reaction mixture was stirred overnight and then
poured on ice H2O. The precipitate was filtered off and dried to af-
ford the title compound as a yellow solid (1.21 g, 94% theoretical
yield).
MS m/z 590.60 (MÀ1, 0.05%), 206.15 (59.35%), 164.15 (43.98%),
57.15 (100%), 41.10 (32.77%). 1H NMR (DMSO-d6): d 1.32 (s, 9H,
CH3), d 2.64–2.82 (m, 8H) d 3.56 (s, 2H, CH2), d 3.70 (s, 6H,
OCH3), d 3.93 (s, 3H, OCH3), d 6.61–6.68 (m, 2H), d 7.23 (d, 2H,
J = 8.5 Hz), d 7.56 (d, J = 8.5 Hz), d 7.61 (s, 1H), d 7.84 (s, 1H), d
10.6 (s, 1H). 13C NMR (DMSO-d6): d 26.7 (CH3), d 28.2 (CH2), d
32.4 (CH2), d 50.5 (CH2), d 55.0 (CH2), d 55.4 (OCH3), d 55.6
(OCH3), d 57.0 (OCH3), d 59.5 (CH2), d 108.9 (CH), d 110.0 (CH), d
111.8 (CH), d 119.7 (CH), d 123.5 (Cq), d 125.8 (Cq), d 126.5 (Cq),
d 128.9 (CH), d 136.0 (Cq), d 136.7 (Cq), d 142.7 (Cq), d 145.2
(Cq), d 146.9 (Cq), d 147.1 (Cq), d 152.0 (Cq), d 162.6 (CON), d
175.1 (COO).
[
11C]-1, at least in rodents, appears to be superior to that of most
other P-gp PET probes ((R)-[11C]verapamil, [11C]-N-desmethyl-lop-
eramide, [11C]laniquidar, [11C]elacridar).17,26,27,37
Another important factor that comes into play when judging
the utility of [11C]-1 for mapping cerebral P-gp is the possible influ-
ence of species differences. Previous data point to pronounced spe-
cies differences in the P-gp system between rats and humans.15,38
We were able to show that comparable plasma concentrations of 1
had an effect about 10 times smaller on (R)-[11C]verapamil brain
distribution in humans as compared to rats, suggesting that P-gp
expression is higher in the human BBB.14,15 This is also consistent
with the observation that baseline brain distribution volume of
(R)-[11C]verapamil was two times lower in humans than in
rats.14,15 This might in turn mean that [11C]-1, which, based on
in vitro experiments with unlabeled 1, binds to P-gp and is not
transported by it,6,8,24 will give a higher signal in humans than in
rats.
4. Material and methods
4.1. General
4.4. 4-Amino-5-(4-[2-(6,7-dimethoxy-1,2,3,4-tetrahydro-2-
isoquinolinyl)ethyl]anilinocarbonyl)-2-methoxyphenyl
pivalate (5)
All chemicals were purchased from Sigma-Aldrich Chemie
GmbH (Schnelldorf, Germany), Merck (Darmstadt, Germany) or
Apollo Scientific Ltd (Bredbury, UK) at analytical grade and used
without further purification. The dimesylate of 1 was obtained
from Xenova Ltd (Slough, UK). The dual P-gp/BCRP inhibitor elacr-
idar hydrochloride was obtained from Glaxo SmithKline (Research
Triangle Park, NC, USA). For administration, 1 dimesylate was
freshly dissolved on each experimental day in 2.5% (w/v) aqueous
(aq) dextrose solution and injected at a volume of 3 mL/kg. Elacri-
dar hydrochloride was dissolved in 20% aq EtOH and injected at a
To a solution of compound 4 (1.15 g, 1.94 mmol) in EtOAc/
MeOH (1:1, 90 mL), which had been purged with argon, Pd/C cata-
lyst (10%, w/w, 0.12 g) suspended in EtOAc/MeOH (1:1, 15 mL) was
added. Hydrogen gas was introduced into the reaction mixture.
The reaction mixture was stirred under normal pressure until
hydrogen uptake was complete. The mixture was filtered and
evaporated to give the title compound as a white solid (0.94 g,
86% theoretical yield).
volume of 2 mL/kg. [11C]CH4 was produced via the 14N(p, 11C nu-
a)
clear reaction by irradiating nitrogen gas containing 10% hydrogen
MS m/z 561.30 (M+, 0.37%), 207.15 (15.76%), 206.15 (100%),
166.15 (10.91%), 57.10 (17.20%). 1H NMR (DMSO-d6): d 1.29 (s,
9H, CH3), d 2.61–2.82 (m, 8H), d 3.54 (s, 2H, CH2), d 2.68–2.74
(m, 9H, OCH3), d 6.41 (s, 1H), d 6.59–6.71 (m, 4H), d 7.19 (d, 2H,
J = 8.4 Hz), d 7.42 (s, 1H), d 7.57 (d, 2H, J = 8.4 Hz), d 9.72 (s, 1H).
13C NMR (DMSO-d6): d 26.9 (CH3), d 28.3 (CH2), d 32.4 (CH2), d
50.6 (CH2), d 55.1 (CH2), d 55.4 (OCH3), d 55.5 (OCH), d 55.5
(OCH3), d 59.6 (CH2), d 99.2 (CH), d 105.5 (Cq), d 110.0 (CH), d
111.8 (CH), d 120.8 (CH), d 122.0 (CH), d 125.9 (Cq), d 126.6 (Cq),
d 128.5 (CH), d 128.9 (Cq), d 135.3 (Cq), d 137.1 (Cq), d 146.9
(Cq), d 147.1 (Cq), d 150.4 (Cq), d 154.1 (Cq), d 166.7 (CON), d
176.3 (COO).
using a PETtrace cyclotron equipped with a CH4 target system (GE
Healthcare, Uppsala, Sweden).
[ a
11C]CH3I was prepared in
TRACERlab FXC Pro synthesis module (GE Healthcare) and con-
verted into [11C]methyl triflate by passage through a column con-
taining silver-triflate impregnated graphitized carbon.39 1H and 13
C
NMR spectra were recorded on a Bruker Advance DP Â 200 (200
and 50 MHz). Chemical shifts are reported in d units (ppm) relative
to Me4Si line as internal standard (s, bs, d, m, Cq for singlet, broad
singlet, doublet, multiplet and quaternary carbon, respectively)
and J values are reported in Hertz. Mass spectra (MS) were ob-
tained with a Shimadzu (GC-17A; MS-QP5050A) spectrometer.
Purity of compounds 4, 5, 6 and 7 was established by analytical
HPLC confirming a purity >95%.
4.5. O-Pivaloyl-tariquidar (6)
4.2. Animals
3-Quinolinecarboxylic acid (0.26 g, 1.50 mmol) was refluxed in
SOCl2 (4 mL) for 3 h followed by evaporation of excess SOCl2. The
acid chloride was then suspended in THF and slowly added to a
suspension of 5 (0.39 g, 0.69 mmol) in THF containing Et3N
(0.75 mL). The reaction mixture was stirred overnight and then
poured on ice H2O. The precipitate was filtered off and dried to
Adult female Sprague Dawley rats weighing 270–290 g were ob-
tained from Harlan Netherlands (Horst, Netherlands). Female FVB
(wild-type), Mdr1a/b(À/À) Bcrp1(À/À) and Mdr1a/b(À/À)Bcrp1(À/À)
,
mice weighing 25–30 g were purchased from Taconic Inc.
(Germantown, USA). The study was approved by the local Animal

5496
F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
afford the title compound as an off-white solid (0.36 g, 72% theo-
retical yield).
column (3
l
m, 3.00 Â 125 mm, Macherey-Nagel, Düren, Germany)
eluted with CH3CN/MeOH/ammonium acetate buffer (0.2 M, pH
5.0) (385:86:529, v/v/v) at a flow rate of 0.5 mL/min. UV detection
was performed at a wavelength of 227 nm. The retention time of
HRMS (ESI/MS): calcd for
C42H44O7N4H: 717.3288, found:
717.3285. 1H NMR (DMSO-d6): d 1.32 (s, 9H), d 2.73–2.98 (m,
8H), d 3.67, (s, 2H, CH2), d 3.82–3.87 (m, 6H, 2 Â OCH3), d 3.90 (s,
3H, OCH3), d 6.55 (s, 1H), d 6.61 (s, 1H), d 7.24–7.32 (m, 2H), d
7.40 (s, 1H), d 7.56 (d, 2H, J = 8.5 Hz), d 7.61–7.68 (m, 1H), d
7.77–7.88 (m, 1H), d 8.5 (d, 1H, J = 8.2 Hz), d 8.30 (s, 1H), d 8.72
(s, 1H), d 8.74–8.79 (m, 1H), d 9.49–9.55 (m, 1H), 12.77 (s, 1H,
NH). 13C NMR (DMSO-d6): d 26.6 (CH3), d 28.6 (CH2), d 33.4
(CH2), d 51.0 (CH2), d 55.6 (CH2), d 55.9 (OCH3), d 55.9 (OCH3), d
56.2 (OCH3), d 60.1 (CH2), d 105.2 (CH), d 109.4 (CH), d 111.3
(CH), d 112.0 (Cq), d 121.1 (2 Â CH), d 121.3 (CH), d 126.0 (Cq), d
126.3 (Cq), d 126.8 (Cq), d 127.0 (Cq), d 127.4 (CH), d 129.2 (CH),
d 129.4 (2ÂCH), d 131.5 (CH), d 134.3 (Cq), d 135.3 (Cq), d 136.0
(CH), d 137.3 (Cq), d 140.3 (Cq), d 147.2 (Cq), d 148.7 (CH), d
149.3 (Cq), d 154.5 (Cq), d 164.0 (CON), d 166.7 (CON), d 175.1
(COO).
[
11C]-1 was about 12–15 min on this HPLC system.
4.8. Small-animal PET imaging and PET data analysis
Prior to each experiment, the animals were placed into an
induction box and anesthetized with 2.5% isoflurane. When uncon-
scious, the animals were taken from the box and kept under anes-
thesia with 1.5–2% isoflurane administered via a mask during the
whole experiment. The animals were warmed throughout the
whole experiment at around 38 °C. Rats were implanted with cath-
eters into the femoral artery (for blood sampling) and vein (for
administration of [11C]-1 and unlabeled inhibitor). In mice, a lateral
tail vein was used for radiotracer administration. The animals were
positioned in the imaging chamber and [11C]-1 (rats: 97 50 MBq
in a volume of about 0.3 mL corresponding to 0.85 0.44 nmol or
4.6. O-Desmethyl-tariquidar (7)
0.55 0.28
lg of unlabeled 1; mice: 30 8 MBq in a volume of
about 0.1 mL corresponding to 0.26 0.07 nmol or 0.17 0.05
l
g
Compound 6 (0.21 g, 0.29 mmol) was suspended in NH3 (2 M in
EtOH, 30 mL) and stirred at 50 °C for 24 h. The mixture was then
evaporated and the product was purified by column chromatogra-
phy (EtOAc/EtOH, 4:1) to afford the title compound as an off-white
solid (0.10 g, 55% theoretical yield).
of unlabeled 1) was administered as an iv bolus over approxi-
mately 30 s. At the start of radiotracer injection, dynamic PET
imaging was initiated using a microPET Focus220 scanner (Sie-
mens, Medical Solutions, Knoxville, USA).
Rats underwent paired PET scans with [11C]-1, before and after
administration of unlabeled inhibitor (1, 15 mg/kg, n = 3, or elacr-
idar, 5 mg/kg, n = 2) (see study outline in Fig. 1). Unlabeled inhibi-
tor was injected iv over approximately 60 s at 60 min after start of
scan 1, which was followed by a further 90 min of PET data acqui-
sition. Scan 2 was performed at 2 h after injection of unlabeled
HRMS (ESI/MS): calcd for
C37H36N4O6H: 633.2713, found:
633.2715. 1H NMR (DMSO-d6): d 2.58–2.91 (m, 8H), d 3.53 (s, 2H,
CH2), d 3.70 (s, 6H, OCH3), d 3.92 (s, 3H, OCH3), d 6.57–6.71 (m,
2H), d 7.23 (d, 2H, J = 8,5 Hz), d 7,44 (s, 1H), d 7.60–7.76 (m, 3H),
d 7.86–7.98 (m, 1H), d 8.10–8.18 (m, 2H), d 8.21 (s, 1H), d 8.84–
8.92 (m, 1H), d 9.23–9.46 (m, 2H), d 10.32 (s, 1H), d 12.14 (s, 1H).
13C NMR (DMSO-d6): d 28.3 (CH2), d 32.4 (CH2), d 50.5 (CH2), d
55.1 (CH2), d 55.4 (OCH3), d 55.5 (OCH3), d 55.7 (OCH3), d 59.5
(CH2), d 105.5 (CH), d 110.0 (CH), d 111.7 (CH), d 115.2 (Cq), d
115.4 (CH), d 121.0 (CH), d 125.9 (Cq), d 126.5 (Cq), d 126.6 (Cq),
d 127.4 (CH), d 127.5 (Cq), d 127.6 (CH), d 128.7 (CH), d 129.3
(CH), d 131.5 (CH), d 132.1 (Cq), d 135.5 (CH), d 136.2 (Cq), d
136.5 (Cq), d 142.2 (Cq), d 146.9 (Cq), d 147.1 (Cq), d 148.2 (CH),
d 148.6 (Cq), d 150.3 (Cq), d 162.8 (CON), d 167.0 (CON).
inhibitor (Fig. 1). During experiments in rats, 5-lL arterial blood
samples were withdrawn manually with pre-weighted micropi-
pettes from the femoral artery (approximately every 5 s) during
the first 3 min after radiotracer injection, followed by further 10-
lL samples taken at 5, 10, 20, 30, 40, 50, 60, 70, 80 and 90 min (last
three time points for scan 1 only). Moreover, one larger blood sam-
ple (0.6 mL) was collected into a heparinized vial at 20 min (scan 1
and scan 2) after tracer injection in order to determine metabolism
of [11C]-1. At the end of scan 2, the rats were sacrificed. A terminal
blood sample (5 mL) was collected and radiometabolites of [11C]-1
in plasma were determined using a previously described SPE/HPLC
assay.26 For HPLC analysis, the same chromatographic conditions
as described above for the semipreparative HPLC purification of
4.7. [11C]Tariquidar ([11C]-1)
Using a TracerLab FXC Pro synthesis module, [11C]methyl triflate
was bubbled through a solution of 7 (0.3 mg, 0.47
(0.5 mL) containing aq NaOH (0.3 M, 5 L, 1.5
l
mol) in acetone
[
11C]-1 were used.
l
lmol, 3.2 equiv).
Mice (n = 3 per mouse type) underwent single 60-min PET scans
After heating for 4 min at 60 °C, the reaction mixture was cooled
(25 °C), diluted with H2O (0.5 mL) and injected into a built-in HPLC
system. A Chromolith Performance RP 18-e (100–4.6 mm) column
(Merck KGaA, Darmstadt, Germany) was eluted at a flow rate of
5 mL/min with CH3CN/MeOH/ammonium acetate buffer (0.2 M,
pH 5.0) (252:56:692, v/v/v). The HPLC eluate was monitored in ser-
ies for radioactivity and ultraviolet (UV) absorption at a wavelength
of 227 nm. On this system radiolabeling precursor 7 and product
with [11C]-1. A separate group of animals (n = 3 per mouse type),
which did not undergo PET scanning, was injected under isoflu-
rane-anesthesia with
0.06 0.04 nmol or 0.04 0.02
[
11C]-1 (7 4 MBq corresponding to
l
g of unlabeled 1) for blood sam-
pling. At 25 min after radiotracer injection, venous blood was with-
drawn by retro-orbital puncture into pre-weighted micropipettes.
Blood samples from the rats and mice were weighted and counted
for activity in a 1-detector Wallac gamma counter (Perkin Elmer
Instruments, Wellesley, USA), which had been cross-calibrated
with the PET camera. Blood activity data were corrected for radio-
active decay and corrected for injected dose per gram body weight
and expressed as SUV.
PET images were reconstructed by Fourier rebinning followed
by 2-dimensional filtered back projection with a ramp filter. The
standard data correction protocol (normalization, attenuation, de-
cay correction and injection decay correction) was applied to the
data. Whole brain was manually outlined on multiple planes of
the PET summation images using the image analysis software
Amide and TACs, expressed as the SUV, were calculated. For the
mouse data, the SUV values measured with PET in brain tissue of
[
11C]-1 eluted with retention times of 3 min and 6–8 min, respec-
tively. The product fraction was diluted with H2O (100 mL) and
passed over a C18 Sep-Pak Plus cartridge (Waters Corporation,
Milford, USA), which had been pre-activated with EtOH (5 mL)
and H2O (10 mL). The cartridge was then washed with H2O
(10 mL) followed by elution of [11C]-1 from the cartridge with EtOH
(3 mL). The EtOH was then removed by heating at 100 °C under a
stream of argon and the product formulated in a mixture of 0.9%
aq saline/EtOH/polyethylene glycol 300 (50:15:35, v/v/v) at an
approximate concentration of 370 MBq/mL for iv injection into ani-
mals. Radiochemical purity and specific activity of [11C]-1 was
determined by analytical radio-HPLC using a Nucleosil 100–3 C18

F. Bauer et al. / Bioorg. Med. Chem. 18 (2010) 5489–5497
5497
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individual animals at 25 min after radiotracer injection were di-
vided by the mean SUV values in whole blood determined in the
four separate groups of mice (see above) to obtain brain-to-blood
ratios of activity.
For all outcome parameters, differences between groups were
tested with a 2-tailed Student’s t-test. The level of statistical signif-
icance was set to p <0.05.
9. van Vliet, E. A.; van Schaik, R.; Edelbroek, P. M.; Redeker, S.; Aronica, E.;
Wadman, W. J.; Marchi, N.; Vezzani, A.; Gorter, J. A. Epilepsia 2006, 47, 672.
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4.9. In vitro autoradiography
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brain sections (10 l
m) of wild-type, Mdr1a/b(À/À), Bcrp1(À/À) and
Mdr1a/b(À/À)Bcrp1(À/À) mice essentially as described elsewhere.26
16. Kreisl, W. C.; Liow, J. S.; Kimura, N.; Seneca, N.; Zoghbi, S. S.; Morse, C. L.;
Herscovitch, P.; Pike, V. W.; Innis, R. B. J. Nucl. Med. 2010, 51, 559.
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5. Conclusions
[
11C]-1 was synthesized and a first in vivo evaluation performed
using small-animal PET imaging. Our data suggest that [11C]-1
interacts specifically with P-gp and BCRP in the BBB. However, fur-
ther experiments are needed to clarify if [11C]-1 behaves in vivo as
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The research leading to these results has received funding from
the Austrian Science Fund (FWF) project ‘Transmembrane Trans-
porters in Health and Disease’ (SFB F35) and from the European
Community’s Seventh Framework Programme (FP7/2007-2013)
under grant agreement number 201380 (‘Euripides’). The authors
thank Gloria Stundner (AIT), Thomas Filip and Maria Zsebedics
(Seibersdorf Laboratories GmbH) for their skillful help with labora-
tory animal handling. Elacridar hydrochloride was kindly provided
by Glaxo SmithKline (Research Triangle Park, NC, USA) and the
dimesylate of 1 by Xenova Ltd (Slough, UK).
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