Discovery, Radiolabeling, and Evaluation of Subtype-Selective Inhibitors for Positron Emission Tomography Imaging of Brain Phosphodiesterase-4D

Article abstract of DOI:10.1021/acschemneuro.0c00077

We aimed to develop radioligands for PET imaging of brain phosphodiesterase subtype 4D (PDE4D), a potential target for developing cognition enhancing or antidepressive drugs. Exploration of several chemical series gave four leads with high PDE4D inhibitory potency and selectivity, optimal lipophilicity, and good brain uptake. These leads featured alkoxypyridinyl cores. They were successfully labeled with carbon-11 (t_1/2 = 20.4 min) for evaluation with PET in monkey. Whereas two of these radioligands did not provide PDE4D-specific signal in monkey brain, two others, [¹¹C]T1660 and [¹¹C]T1650, provided sizable specific signal, as judged by pharmacological challenge using rolipram or a selective PDE4D inhibitor (BPN14770) and subsequent biomathematical analysis. Specific binding was highest in prefrontal cortex, temporal cortex, and hippocampus, regions that are important for cognitive function. [¹¹C]T1650 was progressed to evaluation in humans with PET, but the output measure of brain enzyme density (V_T) increased with scan duration. This instability over time suggests that radiometabolite(s) were accumulating in the brain. BPN14770 blocked PDE4D uptake in human brain after a single dose, but the percentage occupancy was difficult to estimate because of the unreliability of measuring V_T. Overall, these results show that imaging of PDE4D in primate brain is feasible but that further radioligand refinement is needed, most likely to avoid problematic radiometabolites.

Full text of DOI:10.1021/acschemneuro.0c00077

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Research Article
Discovery, Radiolabeling, and Evaluation of Subtype-Selective
Inhibitors for Positron Emission Tomography Imaging of Brain
Phosphodiesterase-4D
Yuichi Wakabayashi,§ Sanjay Telu,§ Rachel M. Dick, Masahiro Fujita, Maarten Ooms, Cheryl L. Morse,
Jeih-San Liow, Jinsoo S. Hong, Robert L. Gladding, Lester S. Manly, Sami S. Zoghbi, Xuesheng Mo,
Emily C. D’Amato, Janice A. Sindac, Richard A. Nugent, Brian E. Marron, Mark E. Gurney,*
Robert B. Innis, and Victor W. Pike*
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ABSTRACT: We aimed to develop radioligands for PET imaging of brain phosphodiester-
ase subtype 4D (PDE4D), a potential target for developing cognition enhancing or
antidepressive drugs. Exploration of several chemical series gave four leads with high PDE4D
inhibitory potency and selectivity, optimal lipophilicity, and good brain uptake. These leads
featured alkoxypyridinyl cores. They were successfully labeled with carbon-11 (t1/2 = 20.4
min) for evaluation with PET in monkey. Whereas two of these radioligands did not provide
PDE4D-specific signal in monkey brain, two others, [11C]T1660 and [11C]T1650, provided
sizable specific signal, as judged by pharmacological challenge using rolipram or a selective
PDE4D inhibitor (BPN14770) and subsequent biomathematical analysis. Specific binding
was highest in prefrontal cortex, temporal cortex, and hippocampus, regions that are
important for cognitive function. [11C]T1650 was progressed to evaluation in humans with
PET, but the output measure of brain enzyme density (VT) increased with scan duration.
This instability over time suggests that radiometabolite(s) were accumulating in the brain.
BPN14770 blocked PDE4D uptake in human brain after a single dose, but the percentage occupancy was difficult to estimate
because of the unreliability of measuring VT. Overall, these results show that imaging of PDE4D in primate brain is feasible but that
further radioligand refinement is needed, most likely to avoid problematic radiometabolites.
KEYWORDS: Phosphodiesterase-4D (PDE4D), inhibitor, radioligand, PET, carbon-11
PDE4D, which through alternative promoter usage and mRNA
splicing encode multiple protein isoforms that differ in their
cellular localization, transcriptional or post-transcriptional
regulation, and activity as dimers or monomers.9−11 The 4A,
4B, and 4D, but not 4C, subtypes exist in brain.
Much evidence indicates that PDE4 inhibition may have
therapeutic benefit in neuropsychiatric disorders.5 BDNF levels
are decreased in major depression and can be increased by
serotonin-reuptake inhibitors such as fluoxetine and citalopram
through their ability to increase brain levels of cAMP.12,13
PDE4 levels are decreased in major depression,14 and this
decrease is reversed by treatment with a serotonin-reuptake
inhibitor (citalopram).15 These studies have provided mech-
INTRODUCTION
■
Nearly all psychiatric medications influence the action of the
biogenic amines, dopamine or serotonin. These medications
include reuptake inhibitors, inhibitors of biogenic amine
metabolism, and subtype-selective receptor agonists, antago-
nists, and inverse agonists. Biogenic amines signal through
their G-protein coupled receptors, which may in turn activate
or inhibit various forms of adenylate cyclase to increase or
decrease levels of the intracellular second messenger, cAMP.
The spatial and temporal patterning of cAMP signaling is
principally regulated by a family of hydrolytic enzymes known
as phosphodiesterase-4 (PDE4),1−4 a member of a broad
superfamily of 11 types of phosphodiesterases.5 Signaling
through cAMP activates downstream effectors such as cAMP-
dependent protein kinase A (PKA), which in turn phosphor-
ylates a key intracellular regulator, known as cAMP response
element binding protein (CREB).6 CREB phosphorylation is
needed for short- and long-term forms of memory and in turn
regulates transcription of the gene encoding brain-derived
neurotrophic factor (BDNF).7,8 The PDE4 gene family
consists of four subtypes, PDE4A, PDE4B, PDE4C, and
Received: February 10, 2020
Accepted: March 26, 2020
Published: March 26, 2020
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anistic insight into major depression and have suggested novel
avenues for therapeutic intervention, such as through the
inhibition of PDE4. One of the earliest inhibitors of PDE4
subtypes, rolipram, was developed for major depression but
was abandoned due to limited efficacy coupled with nausea
and emesis as major dose-limiting side effects.16 Rolipram
binds to all three PDE4 subtypes in brain. Studies now imply
that selective inhibition of the PDE4D subtype may conserve
antidepressant effect and decrease side-effect liability.17
Genetic variation in PDE4B and PDE4D associates with
biological variation in human cognitive function across
multiple, large genome-wide association studies,18 whereas
genetic linkage studies potentially associate PDE4B gene
variants with schizophrenia.19−22 Therefore, selective PDE4
subtype inhibitors, rather than pan-PDE4 subtype inhibitors,
are now keenly sought as potential therapeutics.23−25
At a few research institutes, PDE4 has been imaged in
human subjects with positron emission tomography (PET)
using the pan-subtype selective radioligand [11C](R)-roli-
pram.14,26,27 To expand on these initial findings, we have
sought to develop PDE4 subtype-selective PET radioligands
for brain imaging in human subjects with potential value for
drug discovery and the investigation of neuropsychiatric
disorders. We now report the discovery of novel PET
radioligands selective for PDE4D that for the first time enable
imaging of the distribution of this enzyme in the living primate
brain.
face of the UCR2 helix that can interact with residues glycine-
612, tyrosine-616, isoleucine-617, and methionine-518 across
the hydrophobic surface of the catalytic domain. Largely
hydrophilic residues are exposed to solvent on the opposite
surface of the regulatory helix. The major challenge in our lead
optimization for PET radioligand development was therefore
to balance hydrophobicity (needed for ligand docking) against
hydrophilicity (to reduce nonspecific brain uptake). Moderate
lipophilicity represented by a logD value of around 2.2 is often
considered desirable in a PET radioligand for imaging a
protein target within brain.28 We also utilized the concept of
multiparameter optimization (MPO) as applied to PET
radioligands in the selection of compounds for synthesis.24
Early chemical series of PDE4D inhibitors that we
considered for PET radioligand development were primarily
neutral compounds based on analogs of 1 and 2 and were
developed sequentially with dihydrobenzofuranyl, phenyl,
pyridinyl, pyrimidinyl, and reverse pyrimidinyl cores (SI,
Figure S1). Chemical optimization focused on lowering clogP
from the high values of 1 and 2 (Figure 1) by introducing a
basic nitrogen into the core or into Ar1 or both, and this helped
improve the PET MPO scores to greater than 3.0 (Table 1).
Chemistry. Scheme 1 outlines the methods used to
synthesize the four PDE4D inhibitors, 3−6, that were selected
for labeling. Compounds 3, 5, and 6 were synthesized in a
straightforward manner from the known compounds 11 and
12.29 A Suzuki reaction with the appropriate Ar1 boronic acid
or boronic ester generated compounds 3, 5, and 6. Synthesis of
4 started with commercially available 13 to prepare the
difluoromethoxy compound 14, which was converted into the
benzyl bromide 15. A Suzuki reaction was then used to
generate 4. Syntheses of the O-desmethyl labeling precursors 7,
9, and 10 and the N-desmethyl precursor, 8, are described in
SI.
RESULTS AND DISCUSSION
■
Ligand Design. We embarked on an extensive program of
chemical optimization for the development of a PDE4D-
selective PET radioligand. Previously, we have reported potent
and selective PDE4D allosteric inhibitors in a methoxyphenyl
series in which the core is decorated with aromatic arms that
we designated as Ar1 and Ar2, as here exemplified in the
inhibitors 1 and 2 (Figure 1).23 The PDE4D allosteric binding
Structure−Activity Relationships and Selection of
Compounds for Radiolabeling. A high proportion (∼35%)
of the more than 160 compounds synthesized were highly
potent against PDE4D (IC50 ≤ 10 nM), showing excellent
control over structure−activity relationship for PDE4D
allosteric inhibition (SI, Figure S1). PDE4D inhibitory potency
decreased to some extent across the structural classes in the
order of their investigation as the lipophilicity (logP) was
generally lowered, but selectivity for PDE4D was maintained.
Potent and selective compounds were advanced to mouse iv
cassette pharmacokinetics, based on PET MPO calculations, in
order to generate a brain uptake value.
Of the compounds meeting the criteria for inhibitory
potency for PDE4D* of IC50 ≤ 10 nM and selectivity over
PDE4B of ≥100-fold, four pyridinyl core compounds, 3−6,
were selected for radiolabeling and evaluation as PET ligands.
In addition to potency and selectivity, the compounds
possessed moderate computed lipophilicities, favorable PET
MPO scores, and acceptable peak uptake (>1 SUV) in mouse
brain (Table 1). Compounds 5 (T1660) and 6 (T1650)
showed brain uptake well above 1 SUV (Table 1). Profiling of
6 against a panel of GPCRs and other binding sites did not
reveal significant off-target binding activity (NIMH Psycho-
active Drug Screening Panel). The single hit with IC50 < 10
μM was the serotonin 5-HT2B receptor (IC50 = 766 nM).
Radiochemistry. Each selected inhibitor was labeled with
carbon-11 by O-methylation or N-methylation of the
respective desmethyl precursor (Table 1, compounds 7−10)
with no-carrier-added [11C]iodomethane, itself produced from
Figure 1. Potent and selective PDE4D inhibitors D159687 (1) and
D158681 (2) reported in Burgin et al.23 A pair of aromatic
substituents, Ar1 and Ar2, project from a methoxyphenyl core (Q).
site is created by the closure of a portion of a regulatory helix,
known as the upstream conserved region 2 (UCR2, ∼78 amino
acids), over the active site. An inhibitor completes a
hydrophobic surface that permits docking by the largely
hydrophobic face of UCR2.23 The key UCR2 residue for
PDE4D allosteric inhibitor selectivity is phenylalanine-276. In
PDE4A−C the corresponding residue is a tyrosine. Phenyl-
alanine-276 fits into a hydrophobic cleft in the active site made
by isoleucine-617, methionine-514, and leucine-560, whereas
residues valine-268, isoleucine-272, and leucine-277 form one
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Table 1. Chemical Properties and PDE4 Inhibitory Potencies of Four Compounds (3−6) Selected for Radiolabeling from the
Indicated Desmethyl Precursors (7−10)
a
LogP and logD7.4 values were calculated with Pallas software; values in parentheses were measured with the radiolabeled compound (see SI for
b
details). Human PDE4D7* contained a mutation of S129D to mimic activation by cAMP-dependent protein kinase A (PKA), whereas PDE4B1
contained the mutation S133D. Brain uptake in mice after intravenous dosing (SUV = standardized uptake value calculated as ([brain Cmax (ng/
c
mL)/dose (mg/kg)/1000]).
a
Scheme 1. Syntheses of Ligands Selected for Radiolabeling
a
Reagents and conditions: (a) for 3 and 5, pyridine-4-boronic acid, Pd(dppf)Cl2, K3PO4, dioxane−water, 90 °C, 2 h; (b) for 6, 1-Boc-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(dppf)Cl2, K3PO4, dioxane−water, 90 °C, 15 h; (c) sodium chlorodifluoroacetate, Cs2CO3,
DMF, 65 °C, 48 h; (d) (3-chlorophenyl)boronic acid, K3PO4, SiliaCat DPP-Pd, dioxane, 85 °C, 4.5 h; (e) mCPBA, DCM, RT, 18 h; (f) (i) TFA,
DCM, reflux, 2 h; (ii) LiOH·H2O, MeOH, RT, 3 h; (g) PBr3, DCM RT, overnight; (h) (1-methyl-1H-pyrazol-4-yl)boronic acid, Pd(dppf)Cl2,
K3PO4, dioxane−water, 90 °C, 12 h.
cyclotron-produced [11C]carbon dioxide (Scheme 2). Each
radioligand was then purified by reversed phase HPLC (SI,
Figures S2A−S6A) and formulated for intravenous injection in
adequate activity for PET imaging in monkey or human (SI,
Table S3). Each radioligand was obtained in greater than 13%
radiochemical yield from [11C]carbon dioxide with high molar
activity (>228 GBq/μmol; SI, Table S3), with high radio-
chemical purity (>99%), and in the absence of contaminating
chemical impurities (SI, Figures S2B−S6B). Each radioligand
was radiochemically stable in formulation media for up to 1 h
at room temperature.
PET Imaging in Monkey. After intravenous administra-
tion of [11C]T2525 ([11C]4) to monkey at baseline,
radioactivity rapidly peaked at a high level in whole brain
(5.09 SUV at 4 min) and then declined to a low level (0.79
SUV) by the end of the scan at 120 min (SI, Figure S7A). To
assess any existence of PDE4 specific uptake, we selected the
PDE4 inhibitor rolipram as a preblocking agent that differs in
chemotype from the radioligand. In the subsequent scanning
session where rolipram (0.1 mg/kg) was administered
intravenously to the same monkey at 5 min before [11C]-
T2525, a similar whole brain time−activity curve was obtained
C
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a
Scheme 2. Labeling of Candidate PDE4D PET Radioligands with Carbon-11
a
Reagents and conditions: (a) H2, Ni, 360 °C; (b) I2, 720 °C; (c) either 1 M TBAH (for 3) or 1 M t-BuOK (3.15 μL, 3.15 μmol; for 4−6) in DMF
(80 μL), RT, 5 min.
Figure 2. Lassen plots for [11C]T1660 and [11C]T1650 in monkey based on total distribution volume (VT) measured by two-tissue compartment
modeling in monkey brain. Target occupancy is given by the slope, while the nondisplaceable tracer volume (VND) is given by the X-axis intercept.
(A) Comparison of Lassen plots for [11C]T1650 and [11C]T1660 in which rolipram was used as a preblocker at 1.0 mg/kg, iv. (B) Effect of
reducing rolipram intravenous preblock dose from 1.0 mg/kg to 0.2 mg/kg on Lassen plot, showing decreased slope (occupancy). (C) Comparison
of Lassen plots for use of rolipram at 1.0 mg/kg (iv) or BPN14770 at 3.0 mg/kg (iv) as preblocking agent for [11C]T1650. Note all Lassen plots
gave similar VND values for [11C]T1650. For the use of BPN as preblocker, binding potential (BPND) for whole brain was estimated to be 1.2.
(SI, Figure S7A). Moreover, the radiometabolite-corrected
arterial input functions for these two PET experiments were
very similar (SI, Figure S7B), as were the measured plasma free
fractions (f P) of 1.4% at baseline and 1.75% under preblock
(SI, Table S2). Compartmental modeling corrects for the
difference in blood flow and exposure of the brain under
baseline and blocked conditions by calculating the total volume
of distribution (VT). Brain data could be fitted with two-tissue
compartmental modeling. However, VT was not appreciably
changed after blockade with rolipram, showing that there was
no appreciable specific binding to PDE4D or other subtypes.
After intravenous administration of [11C]T1953 ([11C]3) to
monkey at baseline, radioactivity rapidly peaked at high levels
(4.32 SUV at 4.5 min) in brain regions such as frontal cortex,
hippocampus, and cerebellum and then declined to a low level
(1.07 SUV) by the end of the scan at 90 min (SI, Figure S8A).
In a subsequent scanning session, where rolipram (0.5 mg/kg)
was administered intravenously to the same monkey at 5 min
before [11C]T1953, similar brain region time−activity curves
were obtained but with somewhat faster washout of radio-
activity (SI, Figure S8A). Moreover, the radiometabolite-
corrected arterial input functions for these two PET experi-
ments differed appreciably, with unchanged radioligand
disappearing less rapidly from plasma in the baseline
experiment (SI, Figure S8B), whereas the measured plasma
free fractions were very similar (1.60% and 1.64%, respectively;
Table S4). VT values and VT/fP values were well derived by
two-tissue compartmental modeling. The fitting revealed
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Figure 3. PET imaging of [11C]T1650 in monkey and human. Time−activity curves for monkey brain at baseline and 2 h after BPN14770 (3 mg/
kg, iv) administration in the same monkey (A). Time−activity curves of parent radioligand in plasma in the same paired monkey experiments (B).
Derived measures of VT across monkey brain regions at baseline and after the preblock with BPN14770 (C). Time−activity curves of human brain
at baseline and at 2 h after taking the first dose (50 mg) of BPN14770 (blocked scan) (D). Corresponding time−activity curves of parent
radioligand in plasma at baseline and at 2 h after taking the first dose of BPN14770 in human (E). Comparison of time−activity curves for parent
radioligand in plasma and radioactivity in brain at baseline (30−120 min). Plasma parent SUV decreased throughout the scan, while brain SUV
remained mostly stable (F).
increases, not decreases, across brain regions following
rolipram pretreatment (data not shown). Therefore, no specific
binding to PDE4D or other subtypes was detected.
V
T(baseline). Occupancy by rolipram in this [11C]T1660 study
was estimated to be 79%.
When dosed in rhesus monkey at baseline, the PET time−
activity curves for [11C]T1650 ([11C]6) showed early and high
radioactivity uptake in whole brain (3.28 SUV at 5 min)
followed by extended washout to a moderately low level (1.39
SUV at 90 min and 1.20 SUV at 120 min (Figure 3A). In a
subsequent scanning session, where the selective PDE4D
inhibitor BPN14770 (3 mg/kg) was administered intra-
venously to the same monkey at 5 min before [11C]T1650,
radioactivity in brain peaked early and higher (3.9 SUV at 4
min) than in the baseline scan but then rapidly declined to
0.93 SUV at 90 min (Figure 3A). In the same experiments,
radioligand concentration decreased rapidly in plasma over the
duration of each scan, and in a similar manner (Figure 3B).
There was no change in the input function (arterial plasma) or
f P between the baseline (6.0%) and blocked scans (6.7%) (SI,
Table S4). VT values and VT/fP values were derived well by
two-tissue compartmental modeling. VT decreased by 37%,
revealing appreciable specific binding (VS) of the radioligand
(Figure 3C). A separate pair of PET experiments with
[11C]T1650, using rolipram (1 mg/kg, iv) as preblocking
agent instead of BPN14770, gave highly comparable brain
time−activity curves (SI, Figure S10A) and time−activity
curves of parent radioligand in plasma (SI, Figure 10B). There
was little change in f P between the baseline and blocked scans
(7.0% and 6.2%, respectively), and VT and VT/f P values
decreased by 48% and 41%, respectively, across whole brain
(SI, Figure S10C).
After intravenous administration of [11C]T1660 ([11C]5) to
a monkey at baseline, radioactivity peaked early in brain at a
high level (3.52 SUV at 5.5 min) and then declined to a
moderately low level (1.58 SUV) at 90 min (SI, Figure S9A).
In a subsequent scanning session, in which rolipram (1.0 mg/
kg) was administered intravenously to the same monkey at 10
min before [11C]T1660, radioactivity in brain peaked earlier
and higher (4.15 SUV at 2.25 min) than in the baseline scan
but then rapidly declined to 0.65 SUV at 85 min (SI, Figure
S9A). Radioactivity and parent radioligand cleared rapidly
from arterial plasma during both scans (SI, Figure S9B). There
was little change in fP between the baseline and blocked scans
(7.8% and 8.5%, respectively; Table S4). VT values and VT/f P
values were well derived by two-tissue compartmental
modeling. Rolipram pretreatment decreased VT values and
VT/f P values by 37% and 45%, respectively, across whole brain.
The highest levels of specific binding (VS), calculated as
differences between VT at baseline and after rolipram
pretreatment, were in frontal cortex, temporal cortex, and
hippocampus. Cerebellum had only a very low level of specific
binding (SI, Figure S9C). PDE4D target occupancy by
rolipram in monkey brain was assessed for [11C]T1660 with
a Lassen plot (Figure 2A).30 The assumptions in a Lassen plot
are (i) that there is a range of brain regions with differing target
density (Bmax), (ii) nonspecific binding (VND) is homogeneous
across brain, and (iii) target occupancy is the same in all
regions. In the Lassen plot, target occupancy is given by the
slope of the specific binding [VT(baseline) − VT(blocked)] versus
Time−activity curves in monkey whole brain and brain
regions, such as PDE4D-rich frontal cortex and PDE4D-poor
cerebellum, were readily fitted to a two-tissue compartmental
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Figure 4. Analysis of PET imaging of whole brain, frontal cortex, and cerebellum with [11C]T1650. (A) Time−activity data fitted using two-tissue
compartmental modeling in monkey brain (fits are solid lines). (B) Apparent total distribution volume (VT) time−stability curves for monkey
brain. (C) Time−activity data fitted using two-tissue compartmental modeling in human brain (fits are solid lines). Note: Accurate quantification
of VT was complicated by the accumulation of radiometabolites in human brain. (D) Apparent total distribution volume (VT) time−stability curves
for human brain. The steady increase in VT over the course of the scan is likely due to the accumulation of radiometabolite(s) both entering and
forming in human brain.
model (Figure 4A). VT values in these regions increased as the
length of scanning period after radioligand injection used in
their estimations was increased. VT values from the initial 60
min of scan data reached 90% of the values from the full 120
min of scan data (Figure 4B).
Coronal parametric PET scans (VT) of monkey brain,
obtained with [11C]T1650, showed contrasting heterogeneous
and relatively homogeneous distributions under baseline
(Figure 5A) and preblocked (Figure 5B) conditions.
PDE4D target occupancies by rolipram and BPN14770 in
monkey brain were assessed with [11C]T1650 using Lassen
plots (Figure 2). Preblocking with 1.0 mg/kg (iv) rolipram
gave 93% occupancy, which decreased to 63% when using a
blocking dose of 0.2 mg/kg (iv) (Figure 2B). Use of
BPN14770 at dose of 3 mg/kg (iv) resulted in 63% occupancy
(Figure 2C). Notably, nondisplaceable binding (BPND
)
estimates from these Lassen plots were very similar,
irrespective of blocking agent or dose, in accord with selective
binding of the radioligand to PDE4D only.
Overall, [11C]T1660 and [11C]T1650 showed quite
comparable PDE4D PET imaging performance in monkey.
Plasma free fractions (fP) were similar and readily measurable
with precision. Each radioligand showed high and comparable
brain entry from plasma. The two radioligands also showed
similar specific-to-nondisplaceable ratios (BPND) based on
both VT and VT/f P. Despite many favorable features, both
radioligands were limited in showing nonasymptotically
increasing VT with increasing duration of PET data, a severe
impediment to robust quantification of PDE4D. Nonetheless,
these studies, for the first time, showed that the distribution of
PDE4D in primate brain could be imaged with PET and that
PDE4D is enriched in brain regions important for cognition,
such as prefrontal cortex, temporal cortex, and hippocampus.
PET Imaging in Human. Our PET studies in monkeys
suggested that one of two radioligands, either [11C]T1650 or
Figure 5. Coronal brain parametric PET images obtained with
[11C]T1650. VT in monkey baseline scan (A) and in scan obtained at
2 h after intravenous administration of BPN14770 (3 mg/kg, iv) (B).
SUV in human brain at baseline (C), and at 2 h after BPN14770
(single 50 mg oral dose) (D). BPN14770 showed a blocking effect in
both monkey and human.
[11C]T1660, warranted investigation in human subjects in the
hope that apparent VT would be more stable in humans than in
monkeys and would permit robust quantification. We could
not readily distinguish the performances of [11C]T1650 and
[11C]T1660 in monkeys, and we somewhat arbitrarily chose
[11C]T1650 for study in human, expecting its results would be
similar to those of [11C]T1660.
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Time Course of Radioactivity in Human Brain and
Radioligand in Human Plasma. After intravenous injection
of [11C]T1650, radioactivity readily entered brain, peaked high
(4.39 SUV) after about 4 min, slowly declined, and then
changed little from about 2 SUV from 30 to 120 min (Figure
3D). To determine whether the peak solely reflected uptake of
radioactivity in brain, vascular subtraction of the brain time−
activity curve was performed, on the assumption that blood
comprises 5% of total brain volume.31 The early peak was
minimally affected by subtracting radioactivity from whole
blood, showing that the measured radioactivity was primarily
in brain and not blood.
Following an initial peak of 7.8 SUV at 3 min, the
concentration of unchanged [11C]T1650 in plasma declined
rapidly for several minutes and then minimally declined from
10 to 120 min (Figure 3E). Several radiometabolites
accumulated in plasma and were separated with HPLC (SI,
Figure S11C). Both [11C]A″, which clearly consists of several
unresolved radiometabolites, and [11C]B″ were less lipophilic
than [11C]T1650, whereas [11C]C″ was more lipophilic. [11C]-
A″ increased in concentration throughout the scan, surpassing
70% of the plasma concentration at 50 min after injection,
whereas [11C]B″ remained at about 5% (SI, Figure S12A).
[11C]C″ was only ever present in negligible amounts.
Unchanged radioligand in plasma was only 30% 8% (n =
2 participants) of all radioactivity at 30 min after injection and
about 5% at 120 min (SI, Figure S12A).
curves, and the time instability of VT were all consistent with
the accumulation of radiometabolites in the brain.34
In Vitro and Ex Vivo Formation of Radiometabolites. For
many PET radioligands, labeling at an O-methyl group, as in
[11C]T1650, is unproblematic because demethylation ulti-
mately leads to [11C]carbon dioxide, which does not cross into
brain.35 Nonetheless, if demethylation is slow relative to other
metabolic processes, this can become problematic as brain-
penetrant radiometabolites may form.36
To evaluate the possibility of radiometabolites of
[11C]T1650 accumulating in brain, the stability of
[11C]T1650 was first examined in vitro in blood and plasma
from both rats and humans. After 30 min of incubation at
room temperature, [11C]T1650 was found to be stable in vitro
in human whole blood (94.6%
0.5%, n = 3 participants),
human plasma (96.0% 0.4%, n = 3 participants), rat whole
blood (97.4%, n = 1 rat), and rat plasma (97.2%, n = 1 rat). In
PET experiments, the analytical radiochromatographic profile
for sampled rat plasma (SI, Figure S11A) and monkey plasma
(SI, Figure S11B) resembled that for plasma sampled from
human (SI, Figure S11C), in terms of radioactive components
but not their proportions. Thus, human and rat possibly have a
similar metabolic pathway for [11C]T1650. However, it should
be cautioned that similar retention times on chromatograms
obtained under the same conditions do not necessarily assure
chemical identity. For ex vivo investigation, three rats were
injected with [11C]T1650 and sacrificed after 30 min.
Radioactivity extracted from rat brain showed a group of
unresolved radiometabolites (SI, Figure S11D; peaks [11C]A
and [11C]B) that were less lipophilic than the parent
radioligand, and a low proportion of a radiometabolite that
was more lipophilic (SI, Figure S11C; peak [11C]C). Brain
The radioactivity in brain washed out much more slowly
than the parent radioligand cleared from plasma. Thus, from
60 to 120 min, radioactivity in brain declined by only 7%,
whereas the concentration of [11C]T1650 in plasma declined
by 71% (Figure 3F). These data suggest that radioactive
species other than the parent radioligand were likely
responsible for the higher radioactivity in brain.
radioactivity consisted of 63.3%
3.0% (n = 3) parent
radioligand at 30 min after intravenous injection. To determine
whether any of these radiometabolites were generated within
the brain, the radioligand was incubated with rat brain
homogenates. The radiometabolite group [11C]A, but not
radiometabolite [11C]B, was generated in the brain (SI, Figure
S11E). The presence of radiometabolites in the ex vivo rat
brain seems due not only to the biotransformation of
[11C]T1650 inside the brain itself to [11C]A but also to
radiometabolites ([11C]B) entering brain from periphery.
Therefore, the accumulation of radiometabolites in human
brain is a highly plausible explanation for VT instability.
Human Enzyme Occupancy by BPN14770. To determine
the specificity of [11C]T1650 binding in the human brain, the
blocking effect of the PDE4D-selective therapeutic candidate
BPN14770 was measured. BPN14770 had a clear blocking
effect, whether measured by concentration of radioactivity in
brain or by VT; SUV60−120 decreased by 30%, and VT decreased
by 33% (SI, Figure S13). BPN14770 had virtually no effect on
the time course of the emergence of radiometabolites in
plasma (SI, compare Figure S12, panels A and B). The
decrease of SUV60−120 and VT varied among brain regions, with
the greatest decrease observed in the hippocampus and
amygdala and with a minimal decrease observed in the basal
ganglia, thalamus, and cerebellum.
Pharmacokinetic Modeling of VT in Human Brain. We
used a two-tissue compartment model, which provided fitting
close to the actual time−activity curve for [11C]T1650 (Figure
4C). However, this model overestimated the actual uptake of
radioactivity from 30 to 60 min and underestimated the
radioactivity thereafter. The underestimation of the model at
later intervals may have reflected the accumulation of
radiometabolites in the brain. Taking the limitations of the
modeling into account, VT values for both participants were
highest in temporal cortex (9.63 1.53), occipital cortex (9.52
1.10), and frontal cortex (9.38
cerebellum.
0.76) and lowest in
To determine whether the parent radioligand reached
equilibrium with binding in the brain, VT was calculated for
increasingly truncated portions of the scan, from 0−120 to
0−20 min. The apparent value of VT increased linearly from 20
to 120 min, with a 30% increase over the last 50 min (Figure
4D). The radioligand therefore performed much worse in
humans than in monkeys with regards to quantification of
PDE4D.
Although unstable VT values can have many causes, the two
most common are exceptionally high affinity of the radioligand
causing slow equilibration and accumulation of radiometabo-
lites in the brain.32 T1650 is a potent PDE4D inhibitor, which
presumably reflects a nanomolar binding affinity. Such high
affinity is common for neuroreceptor radioligands33 and does
not usually cause slow equilibration. The slow washout of
radioactivity from human brain, the inadequacy of standard
two-tissue compartment modeling for estimating time−activity
Because quantification in the brain was not robust, possibly
due to the accumulation of radiometabolites, [11C]T1650
could not reliably measure differences in PDE4D density
between participants. We are uncertain how to correct values
such as VT that measure enzyme density in a single participant
for metabolism and cannot easily determine whether this
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the following methods: Method A, Agilent 1100 series, Kinetex EVO
C18 column (2.6 μm, 100 Å, 50 mm × 4.6 mm; Phenomenex), 5−
100% ACN/water (0.07% H3PO4) (3.5 min), 100% ACN (0.07%
H3PO4) (0.25 min), 100%−0% ACN/water (0.07% H3PO4) (0.01
min), 0−5% ACN/water (0.07% H3PO4) (0.29 min), and 5% ACN/
water (0.07% H3PO4); flow rate, 1.0 mL/min. Method B: Agilent
1100 series, XBridge BEH C18 column (3.5 μm, 130 Å, 50 mm × 4.6
mm), 1%−95% ACN/water (0.1% formic acid) (2.0 min), 95%
ACN/water (0.1% formic acid) (2.0 min), 95%−1% ACN/water (1%
formic acid) (0.1 min), 1% ACN/water (1% formic acid) (3.4 min);
flow rate, 1.0 mL/min. Unless otherwise stated, compounds were
≥95% pure by HPLC.
Preparation of 2-(3-Chlorophenyl)-3-methoxy-6-[(pyridin-
4-yl)methyl]pyridine (3; T1953). In a 1 dram vial, 6-(bromo-
methyl)-2-(3-chlorophenyl)-3-methoxypyridine (11; 34 mg, 0.11
mmol) was dissolved in 1,4-dioxane/water (2:1, 1 mL) and then
treated with pyridine-4-boronic acid (30 mg, 0.24 mmol), K3PO4 (50
mg, 0.23 mmol), and Pd(dppf)Cl2 (10 mg, 0.014 mmol). Argon was
bubbled through the solution for 5 min, and then the flask was sealed
under argon and heated to 90 °C for 2 h. The reaction mixture was
concentrated under reduced pressure, and the residue was partitioned
between ethyl acetate and water. The layers were separated, and the
organic phase was dried with Na2SO4 and concentrated. The crude
product was purified by silica gel chromatography using 40% ethyl
acetate/hexanes to give 3 as a white solid: 6 mg (0.020 mmol, 18%);
1H NMR (DMSO-d6, 400 MHz) δ 8.83 (d, J = 6.6 Hz, 2H), 8.00 (d, J
metabolism differs between participants or disease states. It is
hypothetically possible that [11C]T1650 could be used for
repeat studies in a single individual, for example, to measure
enzyme occupancy by a therapeutic candidate. A single 50 mg
oral dose of BPN14770 clearly decreased radioactivity in the
brain (measured as SUV60−120). This result is consistent with
selective binding of the radioligand to PDE4D, because
BPN14770 is highly selective for the D subtype of PDE4.38
However, based on our blocking experiments with BPN14770,
we conclude that [11C]T1650 is unsuitable for measuring
enzyme occupancy due to the difficulty of obtaining reliable
quantification. We do not plan to test [11C]T1660 in humans
because similar performances in monkey plus close structural
similarities suggest that both radioligands would very likely
have similar problems in human.
CONCLUSIONS
■
PET imaging with [11C]T1660 and [11C]1650 selectively
revealed the distribution of PDE4D in monkey brain. This
distribution displayed high specific binding in regions
associated with cognition and relatively low uptake in
cerebellum.
Quantitation of PDE4D in human brain using [11C]T1650
was significantly flawed. Brain uptake could not be explained
using only the parent radioligand in plasma as input for
biomathematical modeling. In retrospect, we now recognize
that this problem was foreshadowed by the time instability of
VT in monkey, which became even worse in humans.
Radiometabolite(s) were likely the cause of this time
instability. The suspected accumulation of radiometabolites
in human brain was supported by the finding of [11C]T1650
radiometabolites in rat brain ex vivo. Nevertheless, a
considerable but indeterminate portion of [11C]T1650 uptake
in human brain was specifically bound to PDE4D because it
was blocked by the PDE4D-selective agent BPN14770. Taking
the limitations of estimating VT for this radioligand into
account, a single 50 mg oral dose of the PDE4D-selective
compound BPN14770 occupied about 40% of the target
enzyme.
= 6.6 Hz, 2H), 7.87 (s, 1H), 7.84 (m, 1H), 7.64 (d, J = 8.6 Hz, 2H),
7.48 (m, 3H), 4.45 (s, 2H), 3.88 (s, 3H); TOF MS ES+ m/z 311.0
[M + H]+; HRMS Calcd for C18H15ClN2O [M + 1]+ 311.0951, found
311.0954; HPLC Method A, tR = 2.57 min.
Preparation of 2-(3-Chlorophenyl)-3-(difluoromethoxy)-6-
[(1-methyl-1H-pyrazol-4-yl)methyl]pyridine (4; T2525). 2-
Bromo-3-(difluoromethoxy)-6-methylpyridine. In a 50 mL round-
bottom flask, 2-bromo-3-hydroxy-6-methylpyridine (13; 4.00 g, 21.3
mmol) was dissolved in DMF (15 mL). To the solution was added
sodium chlorodifluoroacetate (5.2 g, 34 mmol) and Cs2CO3 (14 g, 43
mmol), and the mixture was stirred at 65 °C for 48 h. The reaction
mixture was diluted with water and extracted twice with ethyl acetate.
The combined organic layers were washed twice with brine, dried
with Na2SO4, and concentrated to yield the product: 3.2 g.
2-(3-Chlorophenyl)-3-(difluoromethoxy)-6-methylpyridine (14).
In a 100 mL round-bottom flask, crude 2-bromo-3-(difluorome-
thoxy)-6-methylpyridine (3.00 g, 12.6 mmol) was dissolved in 1,4-
dioxane (40 mL) and then treated with (3-chlorophenyl)boronic acid
(2.36 g, 15.1 mmol), K3PO4 (5.40 g, 25.2 mmol), and SiliaCat DPP-
Pd (0.30 mmol/g, 2.0 g, 0.6 mmol, Silicycle P/N R390-100). The
reaction vessel was purged with argon for 5 min and then sealed and
heated to 85 °C for 4.5 h. The reaction mixture was cooled to RT,
diluted with ethyl acetate, filtered through Celite, and concentrated.
The residue was purified by silica gel chromatography (3% EtOAc/
hexane) to yield 14: 3.47 g (12.7 mmol, 63% over 2 steps).
2-(3-Chlorophenyl)-3-(difluoromethoxy)-6-methylpyridine-N-
oxide. Compound 14 (3.47 g, 12.7 mmol) was dissolved in DCM (40
mL), treated with mCPBA (3.18 g, 14.2 mmol), and stirred at RT for
18 h. Saturated Na2SO3 (2 mL) was added, and the reaction mixture
stirred for 30 min. The phases were separated, and the organic phase
washed with twice with satd. NaHCO3, then dried with Na2SO4 and
concentrated. The unpurified product was used directly in the next
step.
Improved quantitation of PDE4D may well be achieved with
an analog of [11C]T1650 that does not generate radio-
metabolites that accumulate in brain. Overall, this study reveals
that brain PDE4D is a viable target for PET imaging in monkey
and human brain but requires an improved radioligand.
METHODS
■
General. In this article, PDE4D residue numbering is based on the
reference PDE4D7 isoform, GenBank accession No.
NP_001159371.1.
MPO scores were derived according to Zhang et al.24
cLogD and clogP values were computed with Pallas for Windows
software version 3.8 in default option (CompuDrug International; Bal
Harbor, FL).
All radioactivity measurements were corrected for the physical
decay of carbon-11 (t1/2 = 20.4 min).
Results of all statistical analyses are presented as mean SD, unless
otherwise stated as SE (standard error).
Chemistry. Unless otherwise noted, all purchased reagents were
used as received. 1H NMR spectra were recorded on a Bruker Avance
instrument at 400 MHz and obtained in CDCl3 at room temperature
unless otherwise noted. Chemical shifts (δ) were reported in parts per
million (ppm) downfield from the internal standard of tetramethylsi-
lane (0 ppm). Mass spectra were obtained on a Micromass LCT
instrument. Compound HPLC retention times were obtained using
[6-(3-Chlorophenyl)-5-difluoromethoxy-2-pyridyl]methanol. The
crude 2-(3-chlorophenyl)-3-(difluoromethoxy)-6-methylpyridine-N-
oxide was dissolved in DCM (30 mL), treated with TFA (5.6 mL),
and stirred at reflux for 2 h. The mixture was then concentrated to
dryness. The residue was taken up in MeOH (25 mL), treated with
water (1 mL) and LiOH·H2O (600 mg, 14.2 mmol), and then stirred
at RT for 3 h. The volatiles were removed. The residue was diluted
with water and extracted twice with ethyl acetate. The combined
organic layers were dried with Na2SO4 and concentrated to provide
an oil that was used in the next step.
H
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6-(Bromomethyl)-2-(3-chlorophenyl)-3-difluoromethoxypyridine
(15). The crude [6-(3-chlorophenyl)-5-difluoromethoxy-2-pyridyl]-
methanol was dissolved in DCM (20 mL) and cooled to 0 °C. PBr3 (2
mL, 221 mmol) was slowly added, and the reaction mixture was
allowed to warm to RT overnight. The reaction mixture was then
cooled to 0 °C and treated with saturated NaHCO3 (200 mL). The
mixture was extracted 3 times with ethyl acetate, dried with Na2SO4,
and concentrated to give 15: 2.8 g (8.0 mmol, 63% over 4 steps).
2-(3-Chlorophenyl)-3-(difluoromethoxy)-6-[(1-methyl-1H-pyra-
zol-4-yl)methyl]pyridine (4; T2525). In a 25 mL round-bottom flask,
compound 15 (870 mg, 2.5 mmol) was dissolved in 1,4-dioxane/
water (4:1, 7.5 mL) and then treated with (1-methyl-1H-pyrazol-4-
yl)boronic acid (630 mg, 5.0 mmol), K3PO4 (1.06 g, 5.0 mmol), and
Pd(dppf)Cl2 (630 mg, 0.86 mmol). Argon was bubbled through the
solution for 5 min; then the flask was sealed under argon and heated
to 90 °C for 12 h. The reaction mixture was concentrated under
reduced pressure, and the residue was partitioned between ethyl
acetate and water. The layers were separated, and the organic phase
was dried with Na2SO4 and concentrated. The crude product was
purified by silica gel chromatography (100% DCM) to give 4 as an
binding site is identical to those of PDE4A−C. Synthetic genes were
engineered with carboxyl- or amino-terminal hexahistidine tags for
baculovirus-infected insect cell expression and purification. Human
PDE4D7 contained a mutation of S129D to mimic activation by
cAMP-dependent PKA, while PDE4B1 contained the mutation
S133D.37 Both PDE4 proteins contained the mutations S579A and
S581A to remove the potential for ERK-dependent phosphorylation.
Kinetic assay of cAMP hydrolysis by purified PDE4 was measured
by coupling the formation of the PDE4 reaction product, 5′-adenosine
monophosphate, to the oxidation of NADH by the use of three
coupling enzymes (yeast myokinase, pyruvate kinase, and lactate
dehydrogenase), which allowed fluorescent determination of reaction
rates.23 Final concentrations of assay components were as follows: 50
mM Tris, pH 8; 10 mM MgCl2; 50 mM KCl; 2% DMSO; 5 mM
tris(2-carboxyethyl)phosphine; 0.4 mM phosphenolpyruvate; 0.01
mM NADH; 0.04 mM ATP; 0.004 mM cAMP; 7.5 units of
myokinase from yeast; 1.6 units of pyruvate kinase; 2 units of lactate
dehydrogenase; and ∼0.5 nM PDE4D or PDE4B enzymes to yield an
initial rate of about −0.7 RFU/s. All data were normalized relative to
control wells lacking cAMP and are presented as percent inhibition.
An inhibitory concentration 50% (IC50) value was calculated by fitting
a sigmoidal dose response curve. Z′ quality factors were routinely >0.6
for the assay.
Mouse Pharmacokinetic Protocol. Mouse pharmacokinetic
experiments were carried out by AmpliaPharmaTek, Montreal,
Quebec, Canada. Adult male CD1 mice were dosed intravenously
with the test compound dissolved in 5% DMSO, 5% Solutol, and 90%
saline. Up to 3 compounds were dosed as a cassette at 0.1 mg/kg.
Plasma and brain samples were collected (0.083, 0.25, 0.5, 1, 2, 4, 8,
and 24 h) and analyzed by LC-MS/MS. Pharmacokinetic parameters
were determined using WinNonlin. Use of animals was in accordance
with the “NIH Guide for the Care and Use of Laboratory Animals”
(revised 2011) and was approved by the Institutional Animal Care
and Use Committee of the contracting research organization
(AmpliaPharmaTek, Montreal, Quebec, Canada). The mouse brain
SUV was calculated as [brain Cmax (ng/mL)/dose (mg/kg)/1000].
Radiochemistry. Radiochemistry was performed in lead-shielded
hot-cells with automated radiosynthesis apparatus for personnel
protection from radiation. γ-Radioactivity from carbon-11 was
measured with a calibrated dose calibrator (Atomlab 300, Biodex
Medical Systems, Shirley, NY) or an automatic γ-counter (Wizard 3″,
1480 instrument; PerkinElmer; Waltham, MA).
1
oil: 180 mg (0.51 mmol, 21%); H NMR (CDCl3, 400 MHz) δ 7.89
(m, 1H), 7.78 (m, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.42 (m, 3H), 7.29
(m, 2H), 6.21 (t, J = 73.2 Hz, 1H), 4.01 (s, 2H), 3.90 (s, 3H); TOF
MS ES+ m/z 350.2 [M + H]+; HRMS (ES+) Calcd for
C17H14ClF2N3O [M + 1]+ 350.0872, found 350.0873; HPLC Method
A, tR = 3.63 min.
Preparation of 3-Methoxy-2-(3-nitrophenyl)-6-[(pyridin-4-
yl)methyl]pyridine (5; T1660). In a 25 mL round-bottom flask, 6-
(bromomethyl)-2-(3-nitrophenyl)-3-methoxypyridine (12; 164 mg,
0.51 mmol) was dissolved in 1,4-dioxane/water (2.5:1, 7 mL) and
then treated with pyridine-4-boronic acid (112 mg, 0.91 mmol),
K3PO4 (220 mg, 1.04 mmol), and Pd(dppf)Cl2 (45 mg, 0.062 mmol).
Argon was bubbled through the solution for 5 min; then the flask was
sealed under argon and heated to 90 °C for 5 h. The reaction mixture
was concentrated under reduced pressure, and the residue was
partitioned between ethyl acetate and water. The layers were
separated, and the organic phase was dried with Na2SO4 and
concentrated. The crude product was purified by silica gel
chromatography using 6% ACN/DCM to give 5 as a white solid:
1
40 mg (0.13 mmol, 24%); mp, 108.8−109.7 °C; H NMR (CDCl3,
400 MHz) δ 8.92 (t, J = 2.0 Hz, 1H), 8.56 (m, 2H), 8.35 (dt, J = 7.8
Hz, 1.3 Hz, 1H), 8.25 (ddd, 8.2 Hz, 2.2 Hz, 1.0 Hz, 1 H), 7.62 (t, J =
8.0 Hz, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.25 (d, J = 6.0 Hz, 2H), 7.15
(d, J = 8.6 Hz, 1 H), 4.19 (s, 2H), 3.92 (s, 3H); TOF MS ES+ m/z
321.9 [M + H]+; HRMS (ES+) Calcd for C18H15N3O3 [M + 1]+
322.1192, found 322.1195; HPLC method, tR = 2.47 min.
Production of [11C]Carbon Dioxide. [11C]Carbon dioxide (up to
∼85 GBq) was produced with a PETtrace cyclotron (GE Medical
Systems; Milwaukee, WI) according to the 14N(p,α)11C reaction by
irradiation of nitrogen gas38 (initial pressure, 160 psi; volume, 75 mL)
containing oxygen (1%) with a proton beam (16.5 MeV, 45 μA) for
20 or 40 min for radioligand production for monkey and human PET,
respectively.
Preparation of 2-(3-Nitrophenyl)-3-methoxy-6-[(1H-pyra-
zol-4-yl)methyl]pyridin-3-ol (6; T1650). In a 50 mL round-
bottom flask, 6-(bromomethyl)-2-(3-nitrophenyl)-3-methoxypyridine
(12; 520 mg, 1.66 mmol) was dissolved in 1,4-dioxane/water (4:1, 20
mL) and then treated with 1-Boc-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (750 mg, 2.55 mmol), K3PO4 (680
mg, 3.2 mmol), and Pd(dppf)Cl2 (130 mg, 0.17 mmol). Argon was
bubbled through the solution for 5 min, and then the flask was sealed
under argon and heated to 90 °C for 15 h. The reaction mixture was
concentrated under reduced pressure, and the residue was partitioned
between ethyl acetate and water. The layers were separated, and the
organic phase was dried with Na2SO4 and concentrated. The crude
product was purified by silica gel chromatography using 50% ethyl
acetate/hexanes to give 6 as a white solid: 230 mg (0.74 mmol, 45%);
mp, 115.7−117.5 °C; 1H NMR (DMSO-d6, 400 MHz) δ 8.77 (t, J =
1.9 Hz, 1H), 8.42 (d, J = 7.9, 1.3 Hz, 1H), 8.25 (m, 1 H), 7.76 (t, J =
8.0 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.41 (brd s, 1H), 7.29 (d, J =
8.6 Hz, 1H), 3.96 (s, 2H), 3.88 (s, 3H); TOF MS ES+ m/z 311.1 [M
+ H]+, 352.1 [M + ACN + H]+; HRMS (ES+) Calcd for C16H15N4O3
[M + 1]+ 311.1144, found 322.1149; HPLC Method A, tR = 2.96 min.
PDE4 Enzyme Inhibition Assay. Methods used to generate
synthetic genes for human PDE4 subtypes were as described
previously.23 Compounds were tested for PDE4D inhibition and
counter-screened for selectivity against PDE4B, whose allosteric
Production of [11C]Iodomethane. [11C]Iodomethane was pro-
duced from [11C]carbon dioxide in a PETtrace MeI Process Module
or a TracerLab FXC module (GE Medical Systems, Severna Park,
MD) to produce radioligands for monkey and human PET,
respectively. Thus, at the end of proton irradiation, [11C]carbon
dioxide was delivered to the apparatus through stainless tubing (OD
1/8 in, ID 1/16 in) over 3 min, trapped on molecular sieves (13×),
released for reduction to [11C]methane with hydrogen over nickel at
360 °C, and recirculated over iodine at 720 °C.39 The generated
[11C]iodomethane was trapped on Porapak Q held in the
recirculation path.
Syntheses of Radioligands. Desmethyl-precursor (3 μmol) was
dissolved in DMF (80 μL). Either tetra-butylammonium hydroxide in
MeOH (1 M; 3.15 μL; 1.05 μmol) or potassium tert-butoxide in THF
(1 M; 3.15 μL; 1.05 μmol) was added to this solution and then
injected into the loop of an AutoLoop apparatus (Bioscan;
Washington, DC). [11C]Iodomethane was released into the loop of
the apparatus, which was then kept at room temperature for 5 min.
The radioligand was isolated with reversed phase HPLC (see SI Table
S1 and Figures S2A−S6A for chromatographic conditions, retention
times, and radiochromatograms). After removal of mobile phase, the
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radioligand was formulated for intravenous injection in sterile
ethanol−saline (1:10 v/v) and sterile-filtered (Millex-LG 0.22 μm,
25 mm; Merck Millipore; Burlington, MA). The identity and purity of
each radioligand was confirmed with reversed phase HPLC analysis
(SI, Table S2, Figures S2B−S6B) and by LC-MS (ESI) of associated
carrier. Molar activity data are compiled in SI (Table S3).
Radioligand Lipophilicity (LogD) Determinations. The logD
values of radioligands were measured by partition between n-octanol
and sodium phosphate buffer (pH 7.4), as described previously (SI,
Table S4). The amounts of radioactivity in the n-octanol and buffer
phases were determined with a γ-counter.
Stabilities of Radioligands in Monkey Whole Blood and
Plasma In Vitro. Samples of formulated [11C]3−[11C]6 (∼370 kBq;
7 μL) were incubated with monkey whole blood and plasma (200 μL)
in vitro at room temperature for at least 30 min. Radioactive whole
blood samples were then mixed with distilled water (300 μL) for 30 s
to lyse blood cells. Samples (450 μL) of the lysed cells and the
incubated radioactive plasma samples were each added to ACN (720
μL) for deproteinization and then centrifuged (10000g). The
resultant clear supernatant liquids were analyzed with reversed
phase radio-HPLC on an X-Terra C18 column (10 μm, 7.8 mm ×
300 mm, Waters Corp., Milford, MA), eluted with methanol/water/
Et3N (70:30:0.1; by volume) at 4 mL/min. The precipitates were
measured for radioactivity in a γ-counter to allow calculation of the
extraction efficiency. The extraction efficiencies of all samples were
97% 3% (n = 65). The percentage of unchanged radioligand in the
analyte, as determined by radio-HPLC, was divided by the
radiochemical purity of the radioligand to give the stabilities of the
radioligands in whole blood and plasma.
Analysis of Radiometabolites in Monkey Plasma. To determine
each radiometabolite-corrected arterial input function for brain PET
scans, blood samples (0.5−1 mL each) were drawn from the femoral
artery at 15 s intervals until 120 s, followed by 0.5−4 mL samples at 3,
5, 10, 30, 60, 90, and 120 min. The concentration of parent
radioligand was measured with reversed phase HPLC on an X-Terra
column (7.8 mm × 300 mm,10 μm; Waters Corp.) eluted at 5.0 mL/
min with methanol/water/triethylamine (85:15:0.1, by vol.), after
separating plasma from whole blood, as previously described.43
Monkey PET Data Analysis. Total distribution volume (VT), a sum
of a specific binding component known as the specific volume of
distribution (VS) and a nondisplaceable volume of distribution (VND),
may serve as an index of enzyme density. VT equals the ratio at
equilibrium of the concentration of radioligand in tissue to that in
plasma.44 VT values, uncorrected for radioactivity in blood, were
estimated for different brain regions with two-tissue compartmental
modeling using the PET brain time−activity curves and the measured
radiometabolite-corrected arterial input functions. The temporal
stabilities of VT in whole brain in baseline experiments (n = 3)
were assessed by estimating VT from progressively time-truncated data
sets. Additionally, regional brain VT data were used to generate a
Lassen plot30 to estimate PDE4D occupancy in the predose condition
and to estimate VND. The PET image analysis, including kinetic
modeling, was performed with PMOD 3.704 (PMOD Technologies;
Zurich, Switzerland). VT parametric images were created using a
Logan plot.
Human PET Imaging. Participants. This study was approved by
the Combined Neurosciences Institutional Review Board of the
National Institute of Mental Health (Protocol 19-M-0064;
NCT03861000) and the Radiation Safety Committee of the National
Institutes of Health (authorization 2668), and all participants signed
an informed consent form. The study was performed in compliance
with the Health Insurance Portability and Accountability Act. Two
male healthy volunteers (51 3 years of age) participated in the brain
PET scans. All participants were free of current medical or psychiatric
illnesses as determined by medical history, physical examination,
electrocardiogram, urinalysis, and laboratory blood tests (complete
blood count, serum chemistries, and thyroid function test).
Plasma Free Fraction Determinations. Plasma free fractions
(f P) were determined for radioligands in monkey venous plasma and
in pooled arterial human plasma using ultrafiltration through
membrane filters (Centrifree; Millipore; Burlington, MA), as
previously described.40
PET Experiments in Monkey. PET Scanning. PET experiments
were performed in four rhesus monkeys (Macaca mulatta, 7.3−14.4
kg) in accordance with the Guide for Care and Use of Laboratory
Animals41 and were approved by the Animal Care and Use
Committee of the National Institute of Mental Health. Typically,
the monkey subject was anesthetized with ketamine (10 mg/kg, im)
and then maintained in anesthesia with 1.6% isoflurane and 98.4% O2.
The position of the head was fixed using a stereotaxic frame.
Electrocardiogram, body temperature, and heart and respiration rates
were monitored throughout scanning sessions. A microPET Focus
220 scanner (Siemens Medical Solution; Knoxville, TN) was used to
acquire PET images of the brain for up to 120 min after intravenous
administration of radioligand (0.17−0.37 GBq) with molar activities
in the range of 42−514 GBq/μmol at time of injection. Two PET
scanning sessions were performed in the same monkey on the same
day separated by at least 3 h, the first a baseline experiment using
radioligand alone and then a second usually at 10 min following
administration of a dose of PDE4D blocking agent, either (R,S)-
rolipram (1.0, 0.5, 0.2, or 0.1 mg/kg, iv) or BPN14770, a selective
PDE4D inhibitor (3 mg/kg, iv). Although the different doses of
blocking agents and their timings were expected to change relative
target occupancy, we addressed this variance with Lassen plots to
estimate target occupancy and nondisplaceable binding (BPND). The
doses of rolipram were decreased during the course of this study to
minimize the side effect of emesis.
Monkey PET Image Analysis. PET images were reconstructed
using Fourier rebinning plus two-dimensional filtered back-projection.
Images were co-registered to a standardized monkey MRI template
using SPM8 (The Wellcome Trust Centre for Neuroimaging, UCL,
London, UK). For details on our use of this template, see Shrestha et
al.42 A set of 33 predefined brain regions of interest from the template
were then applied to the co-registered PET image to obtain regional
decay-corrected time−activity curves. Reported whole brain data are
from gray matter. All PET images were corrected for attenuation and
scatter. Radioactivity concentrations were expressed as SUV, which
normalizes for weight and injected activity.
Human PET Study Design. Two healthy participants had two PET
scans each with [11C]T1650. Each subject had their two scans in the
same day: a baseline scan in the morning at 10 a.m. and a blocking
scan at 2 p.m. at 2 h after BPN14770 administration (50 mg po at 12
p.m.).
PET Image Acquisition with [11C]T1650 and Administration of
BPN14770. All scans were performed on a Biograph mCT scanner
(Siemens Healthineers; Erlangen, Germany) in three dimensions. CT
scans for attenuation correction were acquired in two dimensions
before radioligand injection. Following a 1 min intravenous bolus
injection of [11C]T1650, brain dynamic emission scans were obtained
for 120 min in 33 frames. PET images were reconstructed with order
subset expectation maximization (OSEM). The activities of
[11C]T1650 administered for the brain scans were 701
90 MBq
(range, 570−775 MBq; n = 4). The administered masses of T1650
were 59 25 ng/kg (range, 36−92 ng/kg; n = 4). There were no
adverse or clinically detectable pharmacologic effects in either of the
two subjects. No significant changes in vital signs or the results of
laboratory studies or electrocardiograms were observed.
Measurement of Unchanged [11C]T1650 in Human Plasma. To
determine arterial input function for the two brain PET scans, blood
samples were drawn continuously from the radial artery for the first 5
min of the scan using an automatic blood sampling system (PBS-101,
COMECER, Joure, Netherlands) followed by discrete samples drawn
manually for the rest of the scan. The concentration of parent
radioligand was measured with radio-HPLC, as previously
described.43 During each participant’s brain scans, 3 mL arterial
blood samples were drawn at 3, 5, 10, 15, 30, and 45 min, 6 mL
arterial samples were drawn at 60 and 75 min, and 9 mL samples were
drawn at 95 and 120 min.
Human PET Image Processing. Three-dimensional T1-weighted
magnetic resonance images (MRIs) were obtained from both brain
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pubs.acs.org/chemneuro
Research Article
scan participants. For region-of-interest (ROI)-based analysis, PET
images were co-registered to the MRIs. A set of 83 predefined regions
from the N30R83 Maximum Probability Atlas45 derived from the
individual’s MRI scan was applied to the dynamic PET images to
obtain regional time−activity curves. For better representation and
noise reduction, the 83 ROIs were subsequently combined into nine
regions via weighted averaging.
Measurement of Radioactivity Uptake in Human Brain. SUV
and area under the curve (AUC) were calculated between 60 and 120
min (SUV60−120). Total distribution volume (VT) for each region was
calculated using two-tissue compartment modeling. The relative
performance of kinetic models was assessed based on the Akaike
Information Criterion (AIC) or F-test.46 The stability of VT values
over time was evaluated by incrementally decreasing the period of
brain data from 120 to 20 min and calculating the relative VT of the
truncated scan to VT of the full scan.
Jinsoo S. Hong − National Institute of Mental Health, Bethesda,
Maryland 20892-9663, United States
Robert L. Gladding − National Institute of Mental Health,
Bethesda, Maryland 20892-9663, United States
Lester S. Manly − National Institute of Mental Health,
Bethesda, Maryland 20892-9663, United States
Sami S. Zoghbi − National Institute of Mental Health, Bethesda,
Maryland 20892-9663, United States
Xuesheng Mo − Tetra Therapeutics, Grand Rapids, Michigan
49506, United States
Emily C. D’Amato − Tetra Therapeutics, Grand Rapids,
Michigan 49506, United States
Janice A. Sindac − Tetra Therapeutics, Grand Rapids, Michigan
49506, United States
Richard A. Nugent − Tetra Therapeutics, Grand Rapids,
Michigan 49506, United States
Brian E. Marron − Tetra Therapeutics, Grand Rapids, Michigan
49506, United States
Robert B. Innis − National Institute of Mental Health,
Bethesda, Maryland 20892-9663, United States
In Vitro and Ex Vivo Stability of [11C]T1650 in Rat. For in vitro
studies, rat brain was harvested, immediately homogenized, and
diluted 2-fold in saline. Brain homogenates were incubated with
[11C]T1650 (32 nM) for 120 min at 37 °C to simulate imaging
conditions. For ex vivo studies, rats were injected intravenously with
[11C]T1650 in 10% ethanol with respect to 0.9% NaCl solution and
sacrificed via thoracotomy 30 min after injection. Anticoagulated
blood was drawn from the myocardium followed by decapitation and
harvesting of the brain. Brain tissues were then homogenized in
acetonitrile (1.0 mL) and then in water (1 mL). Radio-HPLC was
performed for both studies on an X-terra C18 (10 μm, 7.8 mm × 300
mm, Waters Corp.) with a mobile phase of methanol/water/Et3N
(70:30:0.1; by vol.) and a flow rate of 3.75 mL/min.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00077
Author Contributions
§Y.W. and S.T. have equal authorship priority. B.E.M., E.D’A.,
J.A.S., M.E.G, R.A.N., and X.M. contributed to the design and
synthesis of PDE4D allosteric inhibitors. J.A.S., R.A.N., and
X.M. performed chemical synthesis and analyzed character-
ization data. C.L.M., J.S.H., and S.T. performed radiochemistry
and radioanalysis. L.S.M. and S.S.Z. performed experiments on
tissues and small animals and analyzed radiometabolites. M.F.,
M.O., R.L.G., R.M.D., and Y.W. performed PET imaging
experiments and analyses. M.E.G, R.A.N, R.B.I., S.T., and
V.W.P. contributed to radioligand design. M.E.G., R.B.I., and
V.W.P. conceived the project. V.W.P. supervised radio-
chemistry and mainly coauthored the manuscript with
B.E.M., M.E.G., and R.B.I. All authors contributed to, read,
and approved the manuscript.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00077.
■
sı
Chemical syntheses, PET imaging data, HPLC separa-
tion and analytical methods, radiochromatograms, and
NMR data (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Victor W. Pike − National Institute of Mental Health, Bethesda,
Maryland 20892-9663, United States; orcid.org/0000-
0001-9032-2553; Phone: +1 301 5945986; Email: pikev@
mail.nih.gov
Funding
This work was supported by a research grant from the National
Institutes of Mental Health (grant number MH107077) to
M.E.G. and independently by Tetra Discovery Partners, Inc.,
and by the Intramural Research Program of NIH (NIMH
project numbers ZIAMH002852 and ZIAMH002793).
Mark E. Gurney − Tetra Therapeutics, Grand Rapids, Michigan
49506, United States; orcid.org/0000-0003-4901-3083;
Phone: +1-616-224-0084; Email: mark@
tetratherapeutics.com
Notes
The authors declare the following competing financial
interest(s): B.E.M., E.D'A., J.A.S., M.E.G, R.A.N., and X.M.
are employees of Tetra Discovery Partners, Inc.
Authors
Yuichi Wakabayashi − National Institute of Mental Health,
Bethesda, Maryland 20892-9663, United States
Sanjay Telu − National Institute of Mental Health, Bethesda,
Maryland 20892-9663, United States; orcid.org/0000-
0001-5300-8725
Rachel M. Dick − National Institute of Mental Health,
Bethesda, Maryland 20892-9663, United States
Masahiro Fujita − National Institute of Mental Health,
Bethesda, Maryland 20892-9663, United States
Maarten Ooms − National Institute of Mental Health, Bethesda,
Maryland 20892-9663, United States
ACKNOWLEDGMENTS
■
We thank the NIH Clinical PET Center (Director Dr. Peter
Herscovitch) for radioisotope production, NIDDK for HRMS
measurements (Dr. John Lloyd), and the NIMH Psychoactive
Drug Screening Program (Director Professor Bryan L. Roth)
for in vitro ligand screening.
ABBREVIATIONS
■
ACN, acetonitrile; AIBN, azobis(isobutyronitrile); BBB,
blood−brain barrier; BDNF, brain-derived neurotrophic
factor; CREB, cAMP response element binding protein;
DCM, dichloromethane; cLogD, calculated log to base 10 of
the distribution coefficient of a compound between 1-octanol
Cheryl L. Morse − National Institute of Mental Health,
Bethesda, Maryland 20892-9663, United States
Jeih-San Liow − National Institute of Mental Health, Bethesda,
Maryland 20892-9663, United States
K
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Research Article
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and phosphate buffer at pH 7.4.; cLogP, calculated log to base
10 of the partition coefficient of a compound between 1-
octanol and water; GPCR, G-protein-coupled receptors;
mCPBA, meta-chloroperoxybenzoic acid; MPO, multipara-
meter optimization; PDE4, phosphodiesterase-4; Pd(dppf)Cl2,
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II);
PKA, protein kinase A; SUV, standardized uptake value; TFA,
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