Synthesis and evaluation of [N-methyl-11C]N-desmethyl-loperamide as a new and improved PET radiotracer for imaging P-gp function

Article abstract of DOI:10.1021/jm800510m

[¹¹C]Loperamide has been proposed for imaging P-glycoprotein (P-gp) function with positron emission tomography (PET), but its metabolism to [N-methyl-¹¹C]N-desmethyl-loperamide ([¹¹C]dLop; [ ¹¹C]3) precludes quantification. We considered that [¹¹C]3 might itself be a superior radiotracer for imaging brain P-gp function and therefore aimed to prepare [¹¹C]3 and characterize its efficacy. An amide precursor (2) was synthesized and methylated with [¹¹C] iodomethane to give [¹¹C]3. After administration of [¹¹C]3 to wild-type mice, brain radioactivity uptake was very low. In P-gp (mdr-1a(-/-)) knockout mice, brain uptake of radioactivity at 30 min increased about 3.5-fold by PET measures, and over 7-fold by ex vivo measures. In knockout mice, brain radioactivity was predominantly (90%) unchanged radiotracer. In monkey PET experiments, brain radioactivity uptake was also very low but after P-gp blockade increased more than 7-fold. [¹¹C]3 is an effective new radiotracer for imaging brain P-gp function and, in favor of future successful quantification, appears free of extensive brain-penetrant radiometabolites.

Full text of DOI:10.1021/jm800510m

6034
J. Med. Chem. 2008, 51, 6034–6043
Synthesis and Evaluation of [N-methyl-11C]N-Desmethyl-loperamide as a New and Improved
PET Radiotracer for Imaging P-gp Function
Neva Lazarova, Sami S. Zoghbi, Jinsoo Hong, Nicholas Seneca, Ed Tuan, Robert L. Gladding, Jeih-San Liow, Andrew Taku,
Robert B. Innis, and Victor W. Pike*
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892
ReceiVed May 2, 2008
[11C]Loperamide has been proposed for imaging P-glycoprotein (P-gp) function with positron emission
tomography (PET), but its metabolism to [N-methyl-11C]N-desmethyl-loperamide ([11C]dLop; [11C]3)
precludes quantification. We considered that [11C]3 might itself be a superior radiotracer for imaging brain
P-gp function and therefore aimed to prepare [11C]3 and characterize its efficacy. An amide precursor (2)
was synthesized and methylated with [11C]iodomethane to give [11C]3. After administration of [11C]3 to
wild-type mice, brain radioactivity uptake was very low. In P-gp (mdr-1a(-/-)) knockout mice, brain uptake
of radioactivity at 30 min increased about 3.5-fold by PET measures, and over 7-fold by ex vivo measures.
In knockout mice, brain radioactivity was predominantly (90%) unchanged radiotracer. In monkey PET
experiments, brain radioactivity uptake was also very low but after P-gp blockade increased more than
7-fold. [11C]3 is an effective new radiotracer for imaging brain P-gp function and, in favor of future successful
quantification, appears free of extensive brain-penetrant radiometabolites.
Introduction
P-Glycoprotein (P-gpa) is a protein that functions as an ATP-
dependent efflux pump for a wide range of xenobiotics at the
blood-brain barrier1 and at membrane barriers in several other
organs2 such as small intestine, liver, and kidney. P-gp is also
often highly expressed in tumors.2 Hence, P-gp can be a severe
obstacle to the penetration of established or developmental drugs
into the targeted organ or tumor.3 In neurology, P-gp may, for
Figure 1. Structures of loperamide and N-desmethyl-loperamide (dLop;
3).
example, be an obstacle to the brain penetration of anti-HIV
drugs.4 In oncology, P-gp plays a major role in “multidrug
resistance”,5 which is directly responsible for the failure of a
(e.g., difficult radiosynthesis, low sensitivity, or troublesome
high proportion of attempted chemotherapy. Altered expression
metabolism), which have so far compromised their use for
of P-gp may also contribute to the progression of neurodegen-
sensitive and quantitative assessment of P-gp function in vivo,
erative disorders such as Alzheimer’s disease,6-8 HIV encepha-
especially in human subjects.
litis,9 and Parkinson’s disease.10 Hence, elucidation of the
Loperamide is sold over-the-counter as an antidiarrheal agent
expression and function of P-gp in human subjects in vivo could
that acts through agonism of gut µ-opioid receptors.22 This drug
be of great importance in both drug development and medicine.
is normally without harmful central effects solely because P-gp
Moreover, in the field of developing molecular imaging agents
excludes it from brain.23 We were attracted to [11C]loperamide
for use with positron emission tomography (PET) or single
(Figure 1) as a prospective radiotracer for evaluating brain P-gp
photon emission computed tomography (SPECT), the effect of
function because of its safety in human subjects and apparent
P-gp is also frequently encountered, for example, in limiting
ease of radiosynthesis.19,20 However, our initial investigations
the brain entry of some neuroreceptor radioligands.11,12 These
showed that [11C]loperamide is heavily metabolized.24 Demeth-
same imaging modalities, with radiotracers based on P-gp
ylation25,26 of [11C]loperamide gave [N-methyl-11C]N-desm-
substrates, have been proposed for examining P-gp function in
ethyl-loperamide ([11C]dLop, [11C]3; Figure 1) as a radiome-
vivo (for a review, see ref 13). The most widely examined
tabolite that also behaved as an avid substrate for P-gp.24 The
radiotracers include [11C]colchicine,14 [11C]verapamil,15,16 [11C]-
penetration of [11C]3 into the brain, under conditions in which
daunorubicin,15 [18F]paclitaxel,17 94mTc]sestamibi,18 and [11C]-
[
P-gp is absent or inhibited, thwarts the quantitative analysis of
P-gp function with [11C]loperamide because PET is unable to
distinguish between different radioactive species in the field-
of-view; no quantitation of P-gp function has yet been achieved
with this radiotracer. We considered that [11C]3 itself could be
a superior radiotracer, mainly because its metabolism might be
expected to be less troublesome for eventual quantification of
brain P-gp function. Thus, metabolism of [11C]3 is also expected
to occur by demethylation26 but to lead only to single-carbon
radiometabolites such as [11C]methanol. These radiometabolites
will be oxidized and ultimately expired as [11C]carbon dioxide;
they should not accumulate in tissues accessed by the radiotracer
loperamide19,20 for PET and [99mTc]sestamibi for SPECT.21
Many of these radiotracers suffer from one or more limitations
* To whom correspondence should be addressed. Phone: 301-594-5986.
Fax: 301-480-5112. E-mail: pikev@mail.nih.gov. Address: Molecular
Imaging Branch, National Institute of Mental Health, National Institutes of
Health, Building 10, Room B3 C346A, 10 Center Drive, Bethesda, MD
20892-1103.
a Abbreviations: DCPQ, ((2R)-anti-5-(3-[4-(10,11-dichloromethanod-
ibenzosuber-5-yl)piperazin-1-yl]-2-hydroxypropoxy) quinoline trihydro-
chloride; DIPEA, N,N-di-isopropylethylamine; dLop, N-desmethyl-loper-
amide; PET, positron emission tomography; P-gp, P-glycoprotein; RCY,
decay-corrected radiochemical yield; SA, specific radioactivity; SPECT,
single photon emission computed tomography.
10.1021/jm800510m CCC: $40.75
2008 American Chemical Society
Published on Web 09/11/2008

Synthesis and EValuation of [11C]N-Desmethyl-loperamide
Scheme 1. Synthesis of 2, 3, and Radiotracer [11C]3a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6035
t
a Reagents, conditions, and yields: (i) DIPEA, MeCN, 70 °C, 31 h, yield 69%; (ii) KOH, BuOH, 100 °C, 3 d, yield 37%; (iii) [11C]MeI, KOH, DMSO,
80 °C, 5 min, RCY 18% from [11C]carbon dioxide; (iv) MeI, KOH, DMSO, 80 °C, 24 h, 3%.
to cause difficulty in biomathematical analysis of acquired PET
data. Moreover, as a known metabolite of loperamide, 3 would
be safe to administer to human subjects in tracer doses. This
study describes an effective synthesis of [11C]3 for its safe
intravenous administration (Scheme 1) and the evaluation of
[11C]3 in mice with PET and ex vivo measurements, and in
monkeys with PET. [11C]3 shows highly favorable properties
as a new radiotracer for the potential quantification of brain
P-gp function.
Results
Chemistry. Compound 1 was obtained in 69% yield by
alkylation of 4-(4-chlorophenyl)-4-hydroxylpiperidine with 4-bro-
mo-2,2-diphenylbutyronitrile in the presence of DIPEA (Scheme
1). Slow hydrolysis of 1 with KOH in tBuOH gave the required
precursor 2 in 37% yield. Methylation of 2 with iodomethane
gave 3 in low yield but in an adequate amount to serve as a
chromatographic reference material.
[11C]3 was prepared, ready for intravenous injection, from 2
in 18 ( 2% (n ) 20) isolated RCY (decay-corrected radio-
Figure 2. Chromatograms from the HPLC separation used in the
chemical yield) from cyclotron-produced [11C]carbon dioxide.
radiosynthesis of [11C]3. See Experimental Section for chromatographic
The radiosynthesis required 40 min. The obtained activity of
[11C]3 averaged 1.9 ( 0.8 GBq. Specific radioactivity, decay-
corrected to the end of synthesis, averaged 152 ( 48 GBq/
µmol. Radiochemical purity exceeded 99%, and the product was
radiochemically stable for at least 1 h (by radio-HPLC analysis).
[11C]3 was well separated with HPLC from precursor 2 and
other impurities (Figure 2). Thus, chemical impurities were low
and estimated as <1 nmol per batch by assuming that the
impurities have the same extinction coefficient at 225 nm in
the radio-HPLC analysis.
Computation of cLogP, cLogD, and pKa and Measure-
ment of LogD and Apparent pKa. cLogP and cLogD (at pH
) 7.4) values for 3 were 4.16 and 3.49, respectively. The
measured LogD value of [11C]3 was much lower [2.60 ( 0.04
(n ) 6)]. The measured apparent pKa of 3 was between 7.2 and
7.3, and the computed pKa was 7.96.
conditions.
HT1A,1B,1D,1E,2A-2C, ꢀ1,2, D1,2,5, M1-4, κ- and δ-opiate receptors,
and to the norepinephrine and dopamine transporters. Greater
than 50% inhibition was observed at R1A,2A-C, D4, H1-3, µ-opiate
and σ1,2 receptors, and the serotonin transporter. Corresponding
Ki values (nM) were R1A (9.9), R2A (1.0), R2B (7.0), R2C (2.4),
D4 (1.1), H1 (4.2), H2 (1.7), H3 (9.3), µ-opiate (0.6), σ1 (0.8),
σ2 (1.9), and serotonin transporter (6.2).
PET Imaging of [11C]3 in Mouse Brain. After intravenous
injection of [11C]3 into three wild-type mice, the average brain
uptake of radioactivity measured with PET reached a very low
maximum between 2 and 4 min. In three P-gp knockout mice,
average maximal brain uptake of radioactivity was higher and
occurred between 8 and 20 min. The subsequent decrease in
brain radioactivity from all mice was slow. At 35 min after
radiotracer injection, forebrain radioactivity concentration was
Pharmacological Screen of 3. At 10 µM concentration,
3 was found to cause <50% inhibition of binding to 5-

6036 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
LazaroVa et al.
varied regionally, with the putamen showing the highest uptake
and the frontal cortex the lowest. In this experiment, the ratio
of maximal brain radioactivity to that in the baseline experiment
was about five.
In both baseline and P-gp blocked experiments, the uptake
of radioactivity in the pituitary outside the blood-brain barrier
was very high and similar (Figure 5).
PET images of monkey brain, obtained by summing data
acquired between 20 and 90 min after intravenous injection of
[11C]3 under baseline condition, confirmed uniformly very
low uptake of radioactivity into the brain (Figure 6A) and very
high uptake into the pituitary. By contrast, radioactivity was
taken up into all of the brain in the corresponding preblock
experiment and also seen again in the pituitary (Figure 6B).
In experiments in a single monkey, in which the dose of
DCPQ administered before [11C]3 was varied between 0 and
16 mg/kg, iv, radioactivity uptake, averaged between 25 and
50 min, increased almost linearly with dose of DCPQ across
all inspected brain regions (Figure 7). Brain uptake of radio-
activity varied regionally but quite consistently across all doses
of DCPQ. At the highest dose of DCPQ, regional radioactivity
concentrations increased between 7-fold (in frontal cortex) and
13-fold (in cerebellum) over baseline values.
In the PET experiment in which P-gp was inhibited with
DCPQ and the opiate receptor antagonist, naloxone, was
administered at 30 min after injection of [11C]3, there was again
high early uptake of radioactivity into all examined brain
regions. The naloxone had no effect on the rate of washout of
radioactivity from these brain regions (see Supporting Informa-
tion). The administration of DCPQ at either 8 or 16 mg/kg iv
before injection of [11C]3 had no effect on the uptake of
radioactivity into pituitary. Moreover, the administration of
naloxone at 30 min after radiotracer injection had no effect on
washout of radioactivity from pituitary (see Supporting Informa-
tion). Likewise, administration of either loperamide or 3 at 30
min after [11C]3 in DCPQ-treated monkeys had no discernible
effect on the washout of radioactivity from brain (data not
shown).
Figure 3. Average time-activity curves in the forebrain of three wild-
type and three P-gp knockout mice determined with PET after the
intravenous administration of [11C]3. Key: wild-type mice (O); knockout
mice (b). Error bars indicate SD.
on average 3.6-fold higher in the knockout than in the wild-
type mice (Figure 3).
Measurement of [11C]3 and Radiometabolites in Mouse
Brain and Plasma. Recoveries of radioactivity from brain tissue
and plasma into acetonitrile for analysis were between 87.0 and
97.1% (92.5 ( 2.9%, n ) 18). Radioactive analytes were fully
recovered from the HPLC column.
At 30 min after the administration of [11C]3, the total
radioactivity concentrations found in plasma were low and very
similar between knockout and wild-ype mice (Table 1). By
contrast, total radioactivity concentrations in the forebrains of
knockout mice were more than 7-fold higher than the very low
concentrations found in wild-type mice (Table 1).
Three radiometabolites ([11C]A-[11C]C), all less lipophilic
than [11C]3, were detected in mice plasma and brain tissue
samples at 30 min after radiotracer injection (Figure 4). The
concentrations of unchanged [11C]3 in plasma were on average
very low and similar between knockout mice (1.7% SUV) and
wild-type mice (2.8% SUV) (Table 1). This was also true for
the concentrations of each of the three radiometabolites in
plasma and for the two most polar radiometabolites, [11C]A and
[11C]B, in brain. The least polar radiometabolite, [11C]C,
appeared at an 8-fold higher concentration in knockout mouse
brain than in wild-type mouse brain but only at a very low
absolute level compared to that of [11C]3 (Table 1).
Emergence of Radiometabolites of [11C]3 in Monkey
Plasma. After intravenous injection of [11C]3 into a monkey
under baseline or P-gp blocked conditions, radioactivity con-
centration in whole blood decreased rapidly and at similar rates
(data not shown). The recovery of radioactivity from plasma
into supernatant acetonitrile for radio-HPLC analysis was
efficient; only very low percentages of radioactivity coprecipi-
tated with protein. As in mice, [11C]3 and three less polar
radiometabolites were detected in plasma. The concentration
of unchanged [11C]3 in plasma decreased to half of its initial
value within 2 min and was unaffected by preadministration of
DCPQ. The radiometabolites, [11C]a-c, had similar retention
times to those observed in mouse plasma and brain ([11C]A-C,
tRs ) 2.1, 4.5, and 6.9, min, respectively; Figure 4). Although
not proven, the monkey radiometabolites are likely to be the
same radiochemical species as those in mice. Radiometabolite
[11C]b only ever became a low percentage of radioactivity in
monkey plasma, but radiometabolites [11C]a and [11C]c gradu-
ally increased as a percentage of total radioactivity (Figure 8).
In the baseline and P-gp blocked experiments, the times taken
for plasma radiometabolite activity to equal that of [11C]3 were
very similar (∼45 min). DCPQ had little effect on the rate at
which each radiometabolite emerged in plasma.
On average, [11C]3 was 43.6% of radioactivity in the
forebrains of wild-type mice, while in the knockout mice, this
value increased to 91.3%. The uptake of [11C]3 was 16-fold
higher in forebrains of knockout mice than in those of wild-
type mice (Table 1).
The ratios of [11C]3 concentration in forebrain and to that in
plasma were close to unity for wild-type mice but increased to
over 20 for knockout mice (i.e., 35.7% SUV/1.7% SUV, Table
1).
Finally, the concentrations of [11C]3 and of its radiometabo-
lites, [11C]A-C, in cerebellum were very similar to those of
forebrain in both wild-type and knockout mice (data not shown).
PET Imaging of Monkey Brain with [11C]3. After intra-
venous injection of [11C]3 into a monkey under baseline
conditions, the maximal uptake of radioactivity into brain
regions was low but well retained (Figure 5A). The temporal
cortex showed the highest uptake and the cerebellum the lowest
(data for other cortical regions were intermediate and are not
shown). In the experiment in which DCPQ had been pread-
ministered at a dose of 8 mg/kg to preblock P-gp, radioactivity
was taken up more avidly in all measured brain regions, reaching
maxima within 30 min (Figure 5B). Brain uptake of radioactivity
Discussion
The preparation of [11C]3, required a convenient synthesis
of the primary amide 1, a compound which, as far as we are

Synthesis and EValuation of [11C]N-Desmethyl-loperamide
Table 1. Concentration of [11C]3 and its Three Radiometabolites, [11C]A-[11C]C, in P-gp Knockout (KO) and Wild-Type (WT) Mice
forebrain plasma
concentration (%SUV)a concentration (%SUV)a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6037
radiochemical
species
KO
WT
ratio KO/WT
KO
WT
ratio KO/WT
[
[
[
[
11C]A
11C]B
11C]C
11C]3
1.3 ( 0.5
0.3 ( 0.2
1.6 ( 0.8
35.7 ( 9.9
38.9 ( 9.5
2.2 ( 0.7
0.7 ( 0.3
0.2 ( 0.1
2.3 ( 0.2
5.4 ( 1.2
0.6
0.4
8.0
16
12.5 ( 5.7
7.8 ( 7.7
0.1 ( 0.1
1.7 ( 0.3
22.1 ( 5.3
16.9 ( 1.8
5.5 ( 4.1
0.1 ( 0.1
2.8 ( 0.2
25.3 ( 2.6
0.7
1.4
1
0.6
0.9
Total
7
a Data are mean ( SD. Three P-gp knockout and 3 wild-type mice were killed 30 min after intravenous injection of [11C]3.
The uptake of radioactivity at 30 min was about 3.5-fold higher
in forebrains of P-gp knockout mice than those of wild-type
mice (Figure 3). The cerebellum gave similar results (data not
shown). Hence, [11C]3 is a substrate for P-gp, in accord with
the prediction from our earlier study.24
PET scans are unable to identify the chemical species being
measured in the brain. Moreover, they are subject to partial
volume effects due to the limited spatial resolution of the PET
camera. In these experiments, the spatial resolution was ∼1.6
mm full width at half-maximum; therefore, small regions of
mouse brain that contained relatively high levels of radioactivity
would not be measured accurately. They would be underesti-
mated while any neighboring regions of low activity would be
overestimated. To measure radioactivity concentration in brain
and plasma more accurately, analytical measurements were
made ex vivo at a single time point with a γ-counter. The time
chosen for these measurements was 30 min after [11C]3 injection
because the PET scans had already shown little loss of activity
from brain over the preceding time span (Figure 3). Radio-HPLC
of brain tissue (Figure 4) or plasma was also used to separate
and measure unchanged radiotracer and its radiometabolites.
Radio-HPLC of wild-type or P-gp knockout plasma showed
that a high proportion of radioactivity consisted of radiome-
tabolites, all of which were less lipophilic than parent radiotracer.
Measurements in wild-type and knockout mice gave very similar
values for total radioactivity concentration in plasma and for
the distribution of this radioactivity between radiotracer and
radiometabolites (Table 1).
Measurements on brain tissue confirmed the higher radioac-
tivity content in the knockout mice. The ratio of radioactivity
in knockout mice forebrain to that in wild-type mice was 7
compared to the value of about 3.5 seen between 27.5 and 35
min in the PET experiments. The PET ratio is therefore in
appreciable error.
Blood constitutes 4-5% of brain volume. The PET scans
are uncorrected for blood radioactivity, while the ex vivo
measures do not include significant blood radioactivity. In this
case, blood radioactivity was not a major source of error in the
PET measurements because the blood levels of radioactivity
were only about 5-fold higher than in the brain (in wild-type
mice) or appreciably lower (in knockout mice) (Table 1).
Figure 4. Radiochromatogram of radioactive species in P-gp knockout
mouse forebrain at 30 min after the intravenous administration of [11C]3.
See Experimental Section for chromatographic conditions.
aware, has only been mentioned twice in the literature but
without synthesis details.20,27 The synthesis of 1 was ac-
complished by a new route in two steps from commercially
available materials. Slow hydrolysis of 1 to 2 with potassium
t
hydroxide in butanol proved to be a key step; attempts to
achieve this step with a multitude of other reagents were
unsuccessful. A small quantity of reference 3 was obtained by
alkylation of 2 with iodomethane.
[11C]3 was readily prepared for intravenous injection by
alkylation of 2 with [11C]iodomethane, itself prepared from
cyclotron-produced [11C]carbon dioxide. The whole radiosyn-
thesis was performed in a lead-shielded hot-cell with automated
apparatus. Purification by reverse phase HPLC gave [11C]3 in
high radiochemical and chemical purity and high specific
radioactivity. The level of specific radioactivity is unlikely to
be critical for this radiotracer because it is not targeted at
imaging a saturable binding site. The formulated radiotracer was
radiochemically stable.
The measured LogD value of [11C]3 was found to be 2.60
and appreciably different from the computed value (3.49). The
measured value lies in the range normally considered favorable
for good penetration of the blood-brain barrier in the absence
of any effect of efflux transporters.28,29
Loperamide has high affinity for µ-opiate receptors.30
A
pharmacological screen found that 3 had high affinity for
µ-opiate receptors (Ki ) 0.56 nM) and also quite high affinity
(Ki ) <10 nM) for R1A,,2A-2C, D4, H1,3, and σ1,2 receptors, and
for the serotonin transporter, but was devoid of high affinity
for any of a wide battery of other receptors, transporters, and
binding sites. This pharmacological profile is similar to that of
loperamide itself.
The several-fold higher uptake of radioactivity in the fore-
brains of knockout mice compared to those of wild-type is
predominantly explained by the greatly increased uptake of
unchanged [11C]3. In the knockout mice forebrain, uptake of
[11C]3 increased about 16-fold over that in the wild-type mice
forebrain. As a result, about 90% of the radioactivity in knockout
mouse brain was unchanged [11C]3 (Table 1). This result,
obtained in a generally highly metabolic species, augers well
for the potential to quantify brain P-gp function with [11C]3
and PET in rodents and higher species, including humans. By
contrast, we have previously shown that only ∼50% of the
radioactivity in P-gp knockout mouse brain after the administra-
PET scans of wild-type mice administered with [11C]3 alone
revealed only very low uptake of radioactivity into the forebrain,
which quickly maximized and then washed out slowly (Figure
3). These data are consistent with effective exclusion of the
radiotracer from the brain by P-gp at the blood-brain barrier.

6038 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
LazaroVa et al.
Figure 5. Regional uptake of radioactivity in monkey brain after the administration of [11C]3 under baseline conditions (A), and at 30 min after
the intravenous administration of DCPQ (8 mg/kg, iv) (B). Key: temporal cortex (O), cerebellum (0), putamen ( ×), and pituitary (b). In (A), data
for the putamen lie between those of the temporal cortex and the cerebellum (not shown for figure clarity). Frontal and parietal cortical regions gave
curves similar to those of the temporal cortex under each condition.
Figure 6. Summed transaxial PET images of the head obtained between 20 and 120 min after the intravenous administration of [11C]3 to a
monkey under baseline conditions (A) and after P-gp inhibition with DCPQ (8 mg/kg, iv) (B).
Figure 7. Regional brain uptake of radioactivity after intravenous
administration of [11C]3 to a monkey: dependence on preadministered
dose of DCPQ. Key: frontal cortex (9), anterior cingulate (2),
hippocampus (b), occipital cortex (0), and cerebellum (3). Temporal
cortex, parietal cortex, and putamen gave intermediate curves but are
not shown for figure clarity.
tion of [11C]loperamide is unchanged radiotracer, so precluding
prospects for its use to quantify P-gp function.24 Hence, on the
basis of these mice data, [11C]3 is a vastly better radiotracer
than [11C]loperamide.
Encouraged by these results, we pursued PET experiments
in a monkey in which DCPQ at doses equal to or greater than
8 mg/kg iv could be used to block brain P-gp.24 Structurally,
DCPQ is very closely related to zosuquidar ((2R)-anti-5-(3-[4-
(10,11-difluoromethanodibenzosuber-5-yl)piperazin-1-yl]-2-hy-
droxypropoxy)quinoline trihydrochloride; LY335979) (Figure
9), which shows selectivity for P-gp versus other efflux
transporters such as MRP1, MRP2, MRP3, or BCRP.31,32
In a baseline PET experiment with [11C]3 in a monkey, the
uptake of radioactivity in all examined brain regions was very
Figure 8. Time course of composition of radioactivity in plasma after
intravenous administration of [11C]3 into monkey. Key: [11C]3 (b),
[
11C]A (4), [11C]B (3), [11C]C (]], unextracted for analysis (0).
low (Figure 5A). By contrast, uptake of radioactivity into the
pituitary was high. In an experiment in the same monkey in
which DCPQ (8 mg/kg) was administered intravenously before
[11C]3, radioactivity uptake into all brain regions increased
dramatically, while the uptake into pituitary was similar to that
in the baseline experiment (Figure 5B). PET images of the brain
and pituitary under baseline and P-gp blocked conditions
dramatically portrayed these patterns (Figure 6). These data are
consistent with [11C]3 behaving as a substrate for P-gp at the
blood-brain barrier but not at the pituitary, which partially lies
outside the blood-brain barrier.
Regional brain uptakes of radioactivity after intravenous
administration of [11C]3, subsequent to various doses of DCPQ
(0-16 mg/kg, iv) were measured in one monkey and increased
almost linearly with dose of DCPQ (Figure 7). This strongly

Synthesis and EValuation of [11C]N-Desmethyl-loperamide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6039
Three radiometabolites were found in monkey blood after
administration of [11C]3. These were all less lipophilic than
[11C]3 and had similar retention times to those seen in mouse
plasma. Therefore, mouse and monkey likely produce the same
radiometabolites. On the basis of metabolites of 3 identified in
two previous studies,25,26 we hypothesize that these radiome-
tabolites include ring hydroxylated species. [11C]B is only
slightly less lipophilic than [11C]3 and is possibly such a species.
The pattern for the emergence of the radiometabolites in monkey
plasma is shown in Figure 8 and was unaffected by DCPQ
pretreatment. The time taken for half the radioactivity in plasma
to be represented by radiometabolites was about 45 min
(significantly slower than for [11C]loperamide24).
Figure 9. Structures of DCPQ and zosuquidar.
Conclusion
indicates that [11C]3 has sensitivity to the degree of blockade
of brain P-gp.
[11C]3 was confirmed to be an avid substrate for brain P-gp
in mouse and monkey. Although [11C]3 was quite rapidly
metabolized to three less polar radiometabolites, radioactivity
uptake into the brain was greatly increased under conditions in
which P-gp was absent or blocked. In P-gp knockout mice, the
vast majority of radioactivity entering the brain was unchanged
[11C]3 and this was in high ratio to its concentration in plasma.
These findings show that [11C]3 is a new radiotracer with
favorable properties for quantifying brain P-gp function with
PET.
The time-activity curves under baseline and P-gp blocked
conditions were characterized by fast initial uptake of radioac-
tivity and then strong retention of radioactivity (Figures 5). In
addition, some regions like the cerebellum consistently showed
higher uptake than others (e.g., frontal cortex) at all doses of
DCPQ (Figures 5B and 7). This variability may have been
caused by regional differences in blood flow or in P-gp activity
itself. These possibilities require future detailed elucidation.
In view of the high affinity shown by 3 for a variety of
receptors and the serotonin transporter, it was considered that
the strong retention of radioactivity in all brain regions might
represent tight binding to one or more of these sites. 3 showed
highest affinity for µ-opiate receptors. Naloxone is a high-affinity
µ-, κ-, and δ-opiate receptor antagonist and has been used
successfully to displace PET radioligands, such as [11C]di-
prenorphine,33,34 [11C](R)-methyl 4-[(3,4-dichlorophenyl)ace-
tyl]-3-[(1-pyrrolidinyl)methyl]-1-piperazinecarboxylate([11C]-
GR103545),35 and [11C]methylnaltrindole,36 from opiate recep-
tors in a monkey and a human brain in vivo. Therefore, we
attempted to displace radioactivity in brain with an injection of
naloxone at 30 min after the administration of [11C]3 to a P-gp-
inhibited monkey. Naloxone had no discernible effect on the
washout of radioactivity from any of the examined brain regions.
We conclude that specific binding to opiate receptors was not
responsible for the strong retention of radioactivity in brain.
Likewise, naloxone had no effect on washout of radioactivity
from pituitary even though the pituitary is know to contain high
levels of opiate receptors. The uptake of radioactivity into the
pituitary was independent of P-gp inhibition or amount of
inhibitor (DCPQ) administered, showing uptake is not influenced
by P-gp.
Experimental Section
Materials and General Methods. All reagents and organic
solvents were ACS grade or higher and used without further
purification. 4-(4-Chlorophenyl)-4-hydroxypiperidine, 4-bromo-2,2-
diphenylbutyronitrile, and N,N-di-isopropylethylamine (DIPEA)
were purchased from Aldrich (Milwaukee, WI). DCPQ ((2R)-anti-
5-{3-[4-(10,11-dichloromethanodibenzo-suber-5-yl)piperazin-1-yl]-
2-hydroxypropoxy}quinoline trihydrochloride37 was a gift from Eli
Lilly (Indianapolis, IN).
Reactions were performed under argon atmosphere with standard
Schlenk techniques. Yields are recorded for chromatographically
and spectroscopically (1H and 13C NMR) pure materials.
1H (400 MHz) and 13C NMR (100 MHz) spectra of all com-
pounds were recorded on an Avance 400 spectrometer (Bruker;
Billerica, MA). Chemical shifts are reported in δ units (ppm)
downfield relative to the chemical shift for tetramethylsilane.
Abbreviations br, s, d, t, and m denote broad, singlet, doublet, triplet,
and multiplet, respectively.
Mass spectra were obtained on a Polaris-Q GC-MS instrument
(Thermo Fisher Scientific Corp., Waltham, MA). LC-MS was
performed on a LCQ Deca instrument (Thermo Fisher Scientific
Corp.) equipped with a reverse-phase HPLC column (Synergi
Fusion-RP, 4 µm, 150 mm × 2 mm; Phenomenex, Torrance, CA).
The instrument was set up to perform electrospray ionization (spray
voltage 5 kV, nitrogen sheath flow 65 units, auxiliary gas flow 10
units, capillary voltage 35 V, and capillary temperature 260 °C).
For the characterization of synthesized compounds, the column was
eluted at 150 µL/min, either isocratically or with a gradient between
H2O:MeOH:AcOH (90:10:0.5 by vol.) and MeOH:AcOH (100:0.5
v/v). High resolution mass spectra (HRMS) were acquired at the
Mass Spectrometry Laboratory, University of Illinois at Urbana-
Champaign (Urbana, IL) under electron ionization conditions with
a double-focusing high resolution instrument (Autospec; Micromass
Inc.). Samples were introduced through a direct insertion probe.
Thin layer chromatography (TLC) was performed on silica gel
layers (type 60 F254; EMD Chemicals, Gibbstown, NJ), and
compounds were visualized under UV light and by staining with
Dragendorff’s reagent.
We also considered that [11C]3 might have been binding to
one of the other sites for which it has high affinity. The lack of
displacement of radioactivity from monkey brain by loperamide
or 3 showed that [11C]3 does not have saturable receptor binding
in a monkey brain. Most probably, the sustained uptake of
radioactivity in the brain and pituitary represents nonspecific
binding to high-concentration nonsaturable sites or is perhaps
due to some other mechanism. A possibility is entrapment by
protonation because our computation of pKa and experimental
determination of apparent pKa indicate that an appreciable
proportion of 3 will be protonated at physiological pH.
After the intravenous administration of [11C]3, radioactivity
concentration in monkey blood initially decreased rapidly,
reaching a very low and stable radioactivity level at 20 min.
This rate of decrease was unaffected by preadministration of
DCPQ. Likewise, parent radiotracer concentration in blood
decreased fast initially and then continued to decline slowly.
Melting points were measured with a Mel-Temp manual melting
point apparatus (Electrothermal; Fisher Scientific, USA) and were
uncorrected.

6040 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
LazaroVa et al.
[11C]3 and its radiometabolites in samples of biological material
were analyzed on a Nova-Pak C18 column (4 µm, 100 mm × 8
mm; Waters Corp., Milford, MA) housed within a radial compres-
sion module (RCM 100). The column was eluted with MeOH:H2O:
Et3N (70:30:0.1 by vol.) at 2.0 mL/min, with eluate monitored with
a flow through Na(Tl) scintillation detector (Bioscan, Washington,
DC). Methanol (2 mL) was injected onto the column to show no
residual radioactivity after each analysis run. Chromatographic data
were corrected for physical decay to the time of HPLC injection,
stored and analyzed by “Bio-Chrom Lite” software (Bioscan). The
same HPLC method was applied for the determination of radio-
chemical purity, lipophilicity, and radiochemical stability of [11C]3
in various media.
High activities of carbon-11 (>40 kBq, <40 MBq) were
measured with a calibrated ionization chamber (Atomlab 300;
Biodex Medical Systems, Shirley, NY). Low activities of carbon-
11 (<40 kBq) were measured in an automatic γ-counter (model
1480 Wizard; Perkin-Elmer, Boston, MA) with an electronic
window set between 360-1800 keV (counting efficiency, 51.84%).
Measurements of carbon-11 were corrected for any significant
background and for physical decay with a half-life of 20.385 min.38
P-gp knockout mice (mdr-1a(-/-))39 (model 001487-MM,
double homozygotes) and wild-type mice (mdr-1a(+/+)) (model
FVB) were purchased from Taconic Farm (Germantown, NY).
Healthy rhesus monkeys (Macaca mulatta) were used in this study.
All animal experiments were performed in accordance with the
Guide for Care and Use of Laboratory Animals40 and were approved
by the National Institute of Mental Health Animal Care and Use
Committee.
added. The reaction was stirred at 80 °C for 24 h. The crude material
was injected onto a Luna C18 column (10 µm, 10 mm × 250 mm;
Phenomenex, Torrance, CA) eluted at 8 mL/min with aq CF3CO2H
(0.1%):MeCN (72:28 v/v). The collected fractions were then
concentrated under vacuum and repurified on a silica gel rotor
(Chromatotron, model 7924T, Harrison Research, CA) eluted with
ammonium hydroxide solution (2 M) in MeOH:CH2Cl2 (5:95 v/v)
to yield 3 as a pale-yellow solid (16.5 mg, 0.036 mmol, 3.2% yield);
mp ) 224-226 °C (n ) 3). TLC (silica gel; CH2Cl2:2 M NH4OH
in MeOH (95:5 v/v); Rf ) 0.45. 1H NMR (CDCl3): δ 7.43 (d, J )
9.2 Hz, 2 H), 7.30 (m, 12H), 6.62 (s, 1H), 2.80 (d, J ) 4.8 Hz,
3H), 2.66 (t, J ) 7.2 Hz, 2H), 2.39 (m, 4H), 2.08 (t, J ) 11.1 Hz,
2H), 1.72 (d, J ) 11.6 Hz, 2H), 1.60 (d, J ) 21.2 Hz, 2H). 13C
NMR (CDCl3): δ 174.79, 143.76, 132.79, 128.77, 128.42, 128.34,
126.91, 126.09, 70.96, 60.10, 58.47, 55.08, 49.50, 26.68. LC-MS,
(M+ + 1) 463.2. HRMS, found (M+ + 1) 463.2144; calcd for
C28H32ClN2O2, 463.2152. LC: 99.90%.
Pharmacological Screen of 3. 3 was submitted to the National
Institute of Mental Health Psychoactive Drug Screening Program
(NIMH-PDSP) for assessment of binding affinity against a wide
range of receptors and transporters (5-HT1A,1B,1D,1E,2A-C,3,5A,6,7
,
R
1A,2A-2C, ꢀ1,2, µ-, κ-, δ-opiate, D1,2,4,5, H1-3, M1-5, and σ1,2
receptors, and noradrenaline, serotonin, and dopamine transporters).
Detailed assay protocols are available at the NIMH-PDSP Web site
(http://pdsp.cwru.edu).
Production of [11C]Iodomethane. No-carrier-added [11C]carbon
dioxide (∼38 GBq) was produced in a target of nitrogen gas (∼164
psi) containing oxygen (1%) via the 14N(p,R)11C reaction induced
for 20 min with a 16 MeV proton beam (45 µA) from a PETrace
cyclotron (GE; Milwaukee, WI). [11C]Iodomethane was produced
within a lead-shielded hot-cell from the [11C]carbon dioxide via
reduction to [11C]methane and iodination41 within a MeI MicroLab
apparatus (GE).
Group data are expressed as mean ( SD.
4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphe-
nylbutanenitrile (1). 4-(4-Chlorophenyl)-4-hydroxypiperidine (2.12
g, 10.0 mmol) was suspended in acetonitrile (15 mL) and DIPEA
(3.5 mL, 30 mmol) was added. 4-Bromo-2,2-diphenylbutyronitrile
(3.00 g, 10.0 mmol) in acetonitrile (15 mL) was then added. The
reaction mixture was stirred under argon at 70 °C for 31 h. After
concentration under vacuum, the crude material was redissolved
in dichloromethane and introduced onto a silica gel column. The
product was eluted with ammonium hydroxide solution (2 M) in
MeOH:CH2Cl2 (6: 94 v/v) to yield 1 as a pale-orange solid (3.10
g, 7.21 mmol, 69% yield); mp 108-109 °C (n ) 3). TLC (silica
gel; CH2Cl2:2 M NH4OH in MeOH (95:5 v/v); Rf ) 0.60. 1H NMR
(CDCl3): δ 7.36 (m, 14H), 2.76 (d, J ) 11.20 Hz, 2H), 2.65 (m,
4H), 2.48 (t, J ) 6.9 Hz, 2H), 2.08 (t, J ) 12.5 Hz, 2H), 1.68 (d,
J ) 11.58 Hz, 2H), 1.60 (br s, 1H). 13C NMR (CDCl3): δ 140.16,
132.95, 129.09, 128.57, 128.10, 126.95, 126.23, 122.27, 71.11,
54.93, 50.17, 49.71, 38.52, 36.80. LC-MS (M+ + 1) ) 431.2.
HRMS (M+ + 1): found, 431.1895; calcd for C27H27ClN2O,
431.1890. LC: 99.89%.
Preparation of [11C]3. Radiochemistry was performed in a PLC-
controlled semirobotic Synthia apparatus42 (Synthia, Uppsala,
Sweden), housed within the same lead-shielded hot-cell used to
prepare [11C]iodomethane. [11C]Iodomethane in carrier helium (15
mL/min) was bubbled into a sealed 1 mL vial containing 2 (1.0
mg, 2.23 µmol) and KOH (5.0 mg, 89.3 µmol) in DMSO (0.4 mL).
When the radioactivity in the vial had maximized, the reaction
mixture was heated at 80 °C for 5 min and then diluted with water
(500 µL). The crude material was injected onto a Gemini C18
column (5 µm, 10 mm × 250 mm; Phenomenex) eluted at 6 mL/
min with ammonium hydroxide solution (2 M) in MeOH:CH2Cl2
(62:38 v/v). Eluate was monitored for radioactivity (pin diode
detector HC-003; Bioscan) and absorbance at 225 nm (Gold 166
detector; Beckman). [11C]3 (tR ) 10.2 min) eluted after 2 (tR
)
8.71 min) and was collected in a 10 mL round-bottom vial
containing an aqueous solution (0.1 mL) of ascorbic acid (1 mg;
USP grade). This was then rotary evaporated to dryness, diluted
with sterile saline for injection (10 mL; USP grade), and filtered
through a sterile filter (Millex MP, Millipore, Bedford, MA). The
pH of the dose was 4.5.
4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphe-
nylbutanamide (2). Compound 1 (2.50 g, 6.00 mmol) was
t
dissolved in butanol (20 mL) and potassium hydroxide (1.18 g,
21.0 mmol) was added. The reaction mixture was stirred at 100 °C
for 3 d. After concentration under vacuum, the crude material was
redissolved in dichloromethane and filtered through a pad of celite.
Chromatography of the sample on a silica gel column eluted with
ammonium hydroxide (2 M) solution in MeOH:CH2Cl2 (5:95 v/v)
gave 2 as a pale-yellow solid (1.09 g, 2.4 mmol, 37% yield); mp
) 208-210 °C (n ) 3). TLC (silica gel; CH2Cl2:2 M NH4OH in
[11C]3 was analyzed for radiochemical purity on a Luna C18
column (5 µm, 4.6 mm × 250 mm; Phenomenex) eluted with aq
CF3CO2H (0.1%):MeCN (40:60 v/v) at 2.5 mL/min (tR ) 5.55 min),
with eluate monitored for absorbance at 225 nm (Gold 166 detector,
Beckman) and radioactivity (pin diode detector HC-003; Bioscan).
The identity of [11C]3 was confirmed by (i) LC-MS-MS of
associated carrier, and (ii) observation of coelution with added
authentic 3 in a second radio-HPLC analysis.
1
MeOH (95:5 v/v); Rf ) 0.45. H NMR (CDCl3): δ 7.35 (d, J )
4.80 Hz, 2 H), 7.26 (m, 12H), 6.49 (s, 1H), 5.51 (s, 1H), 2.77 (d,
J ) 11.37 Hz, 2H), 2.61 (t, J ) 7.67 Hz, 2H), 2.33 (m, 4H), 2.03
(t, J ) 12.64 Hz, 2H), 1.70 (br s, 1H), 1.64 (d, J ) 11.91 Hz, 2H).
13C NMR (CDCl3): δ 176.56, 143.26 132.82, 128.69, 128.41,
127.06, 126.10, 70.90, 59.91, 54.92, 49.48, 38.28, 35.87. LC-MS
(M+ + 1) 449.2. HRMS (M+ + 1) found, 449.2012; calcd for
C27H30ClN2O2, 449.1996. LC: 99.90%.
Computation of cLogP, cLogD, and pKa and Measurement
of LogD and apparent pKa. cLogP, cLogD (at pH ) 7.4), and
pKa values for 3 were computed with the program Pallas 3.0 for
Windows (CompuDrug; S. San Francisco, CA).
The LogD value of [11C]3 was determined by measuring its
distribution between n-octanol and sodium phosphate buffer (0.15
M, pH 7.4), as previously described for other radiotracers,43,44 and
the apparent pKa determined by the method described previously,
except that extraction was performed by using cyclohexane instead
of 1-octanol.43
4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenyl-
N-methyl-butanamide (3). Compound 2 (0.5 g, 1.12 mmol) was
dissolved in DMSO (3 mL) at 24 °C and then potassium hydroxide
(81.2 mg, 1.45 mmol) and iodomethane (158 mg, 1.1 mmol) were

Synthesis and EValuation of [11C]N-Desmethyl-loperamide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6041
PET Imaging of [11C]3 in Mouse Brain. Brains of three wild-
type and three P-gp knockout mice were scanned with the Advanced
Technology Laboratory Animal Scanner (ATLAS). This small-
animal PET camera has effective transaxial and axial fields of view
of 6.0 and 2.0 cm, respectively.45 Mice were anesthetized with 1.5%
isoflurane in oxygen and body temperatures maintained between
36.5 and 37.0 °C with a heating pad or lamp. Radiotracer was
injected via a polyethylene cannula (PE-10; Becton Dickinson,
Franklin Lakes, NJ) secured in the mouse tail vein with tissue
adhesive (Vetbond; 3M, St. Paul, MN).
On two occasions, three mice (at least one P-gp knockout mouse
(19-23.8 g) and one wild-type mouse (28.6-29.5 g)) were placed in
the camera gantry and each injected with a bolus of [11C]3 (21.1-27.9
MBq; SA 40.7-131 GBq/µmol). The injected radioactivities gave
count rates within the linear range of scanner performance, i.e.,
<300000 singles per s. Scans were obtained from the time of injection
for 100 min in the frame sequence 6 × 20 s, 5 × 1 min, 4 × 2 min,
3 × 5 min, 3 × 10 min, and 2 × 20 min. Data were corrected for
random events and detector efficiency. Images were reconstructed with
a 3D ordered-subset expectation maximization algorithm into 17
coronal slices with 3 iterations and 16 subsets, resulting in a resolution
of about 1.6 mm full width at half-maximum.46,47 The reconstructed
voxel size was 0.56 mm × 0.56 mm × 1.12 mm. No attenuation or
scatter correction was applied.
sterile filter (Anatop 25; 0.2 µm, 25 mm; Whatman). This DCPQ
solution (3.36 mg/mL; 19.76 mL) was infused into the monkey
over 10 min. After 20 min, the monkey was injected with [11C]3
(218 MBq).
Experiments were similarly performed in a single monkey
(monkey B; 15.5 kg) to assess the effect of DCPQ at doses of 0, 4,
8, and 16 mg/kg (iv). on regional brain radioactivity uptake after
the administration of [11C]3. Time-radioactivity data were collected
for the same eight brain regions as previously described. Injected
activities in this series of experiments were 377, 340, 281, and 355
MBq at specific activities of 160, 94.5, 131, and 183 GBq/µmol,
respectively.
The baseline and P-gp blocked experiments were repeated in a third
monkey (C; 12.45 kg) with naloxone (5 mg/kg, iv) administered at
30 min after the second injection of [11C]3, and in another monkey
(D; 15.72 kg) in which the dose of DCPQ was increase to 16 mg/kg
(iv) and naloxone was given as before. Injected activities in this
sequence of experiments were 313, 283, 377, and 355 kBq, respectively.
A PET experiment was performed in monkey E (11.1 kg) in
which P-gp was blocked with DCPQ (8 mg/kg, iv) as before and
in which loperamide (1 mg/kg, iv) was also given at 30 min after
[11C]3 (315 MBq). Finally, This experiment was repeated in
monkey F (9.5 kg), except that 3 (1 mg/kg, iv) was given instead
of loperamide before administration of [11C]3 (370 MBq). This
scan was terminated at 45 min.
Images were analyzed with PMOD (pixel-wise modeling soft-
ware; PMOD Group, Zurich, Switzerland). A region of interest was
drawn for forebrain on coronal slices guided by a mouse brain
stereotaxic atlas.48 Brain uptake of radioactivity was corrected for
decay and normalized for injected dose and body weight by
expression as percent standardized uptake value (%SUV), defined
as: %SUV ) [(activity per g tissue)/injected activity] × g body
weight × 100.
Emergence of Radiometabolites of [11C]3 in Monkey
Plasma. After the administration of [11C]3 to monkey A under
baseline and P-gp blocked condition (achieved with DCPQ at 8
mg/kg, iv), eight arterial blood samples (0.5 mL each) were drawn
into heparin-treated syringes at 15 s intervals until 2 min, followed
by 1 mL aliquots at 3, 5, 10, 20, 30, 45, 60, 75, 90, and (in DCPQ-
treated monkey only) 120 min. Samples were measured for
radioactivity. Plasma [11C]3 was separated, measured for radioac-
tivity, deproteinized, and the [11C]3 and radiometabolite contents
quantified with radio-HPLC (General Method A).
Measurement of [11C]3 and Radiometabolites in Mouse
Plasma and Brain. Thirty minutes after injection of [11C]3 into
each of three wild-type and three knockout mice, anticoagulated
blood (1 mL) was sampled by cardiac puncture. Plasma (∼100-450
µL) was separated by centrifugation, deproteinized with acetonitrile
(700 µL), and measured for radioactivity in an automatic
γ-counter.49 The animals were decapitated, and forebrains and
cerebella removed for immediate radioanalysis.49 Brain-tissue
radioactivities were measured in the γ-counter. Brain tissue
suspension, along with carrier 3, was homogenized in 1.5 times its
volume of acetonitrile with a hand-held tissue Tearor (model 985-
370; BioSpec Products Inc.). Water (500 µL) was added and the
mixture homogenized again. Homogenates were then centrifuged
at 10000g for 1 min. The resulting precipitates and supernatant
liquids were measured for radioactivity to allow the recovery of
activity into the acetonitrile supernatants to be calculated. Aliquots
of the clear prefiltered supernatant liquids were analyzed by radio-
HPLC (see General Methods).
Acknowledgment. This research was supported by Intra-
mural Research Program of the National Institute of Mental
Health (project no. Z01-MH-002795-04). We are grateful to the
NIH Clinical Research Center PET Group for radionuclide
production and for assistance on PET Imaging, to PMOD
Technologies for providing the image analysis software and the
NIMH Psychoactive Drug Screening Program (PDSP) for
performing assays; the PDSP is directed by Bryan L. Roth, MD,
Ph.D. (University of North Carolina at Chapel Hill) with project
officer Jamie Driscol (NIMH) (contract no. NO1MH32004). We
are also grateful to GlaxoSmithKline for a gift of reference 3
and to Eli Lilly for DCPQ.
PET Imaging of Monkey Brain with [11C]3. One male rhesus
monkey (A; 8.4 kg) was fasted overnight, immobilized with ketamine
(10 mg/kg, im), intubated, placed on a ventilator, and anesthetized
with 1.6% isoflurane in O2. Body temperature was maintained between
36.5 and 37.0 °C. After injecting [11C]3 (211 MBq in 10 mL) through
an intravenous perfusion line filled with saline, dynamic PET scans
of the brain were acquired on an HRRT camera (Siemens, Knoxville,
TN) for 120 min in 33 frames of duration increasing from 30 s to 5
min. Images were reconstructed using a list mode 3D-OSEM algo-
rithm,50 resulting in a resolution of 2.5 mm full width at half-maximum.
Scatter and attenuation correction were applied. Images were analyzed
with PMOD. Regions of interest were drawn on coronal slices for
eight brain regions (frontal cortex, anterior cingulate, temporal cortex,
parietal cortex, hippocampus, occipital cortex, putamen, and cerebel-
lum). Activity was decay-corrected to the time of injection and
expressed as %SUV.
Supporting Information Available: HPLC chromatograms
verifying the purities of compounds 1-3 and PET time-activity
data for brain regions in P-gp inhibited monkey given naloxone
after radiotracer [11C]3. This material is available free of charge
via the Internet at http://pubs.acs.org.
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