In vivo imaging of histone deacetylases (HDACs) in the central nervous system and major peripheral organs

Article abstract of DOI:10.1021/jm500872p

Epigenetic enzymes are now targeted to treat the underlying gene expression dysregulation that contribute to disease pathogenesis. Histone deacetylases (HDACs) have shown broad potential in treatments against cancer and emerging data supports their targeting in the context of cardiovascular disease and central nervous system dysfunction. Development of a molecular agent for non-invasive imaging to elucidate the distribution and functional roles of HDACs in humans will accelerate medical research and drug discovery in this domain. Herein, we describe the synthesis and validation of an HDAC imaging agent, [¹¹C]6. Our imaging results demonstrate that this probe has high specificity, good selectivity, and appropriate kinetics and distribution for imaging HDACs in the brain, heart, kidney, pancreas, and spleen. Our findings support the translational potential for [¹¹C]6 for human epigenetic imaging.

Full text of DOI:10.1021/jm500872p

Article
pubs.acs.org/jmc
Open Access on 09/09/2015
In Vivo Imaging of Histone Deacetylases (HDACs) in the Central
Nervous System and Major Peripheral Organs
Changning Wang,† Frederick A. Schroeder,†,‡ Hsiao-Ying Wey,† Ronald Borra,† Florence F. Wagner,§
Surya Reis,‡ Sung Won Kim,∥ Edward B. Holson,§ Stephen J. Haggarty,‡ and Jacob M. Hooker*,†
†Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical
School, 73 High Street, Charlestown, Massachusetts 02129, United States
‡Chemical Neurobiology Laboratory, Departments of Neurology and Psychiatry, Center for Human Genetic Research, Massachusetts
General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States
§Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts
02142, United States
∥Laboratory of Neuroimaging, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: Epigenetic enzymes are now targeted to treat
the underlying gene expression dysregulation that contribute
to disease pathogenesis. Histone deacetylases (HDACs) have
shown broad potential in treatments against cancer and
emerging data supports their targeting in the context of
cardiovascular disease and central nervous system dysfunction.
Development of a molecular agent for non-invasive imaging to
elucidate the distribution and functional roles of HDACs in
humans will accelerate medical research and drug discovery in
this domain. Herein, we describe the synthesis and validation
of an HDAC imaging agent, [11C]6. Our imaging results
demonstrate that this probe has high specificity, good selectivity, and appropriate kinetics and distribution for imaging HDACs in
the brain, heart, kidney, pancreas, and spleen. Our findings support the translational potential for [11C]6 for human epigenetic
imaging.
far to investigate HDAC distribution17,18 are not compatible
with translational medicine. Therefore, our goal was to develop
a positron emission tomography (PET) imaging probe, which
can quantify the density of HDAC 1, 2, 3, and 6 in vivo.
Evaluating the expression and distribution of epigenetic
machinery in vivo will improve our understanding of their
relationship with disease and would provide an opportunity to
intervene with treatment.
INTRODUCTION
■
Eukaryotic cellular identity, growth, and homeostasis, extending
to the level of organs, systems, and whole organisms depends in
part on refined and adaptive regulatory control of gene
expression. Investigation of epigenetic mechanismsnon-
genetic determinants of gene expression variabilityhas
highlighted the role of chromatin modifying enzymes in
integrating environmental cues into biological effects via
changes in transcriptional activity.
The challenge for HDAC research is developing a technique
for quantifying the HDAC density that can be used in living
To date, much attention in this regard has been paid to the
histone deacetylase (HDAC) family of enzymes.1−16 HDAC
subjects. The non-invasive imaging technique, PET, is an
excellent tool to visualize chromatin-modifying enzymes in
enzymes are divided into four different classes based on
animals and human. PET can provide molecular level insight
sequence homology, cellular localization, and phylogenic
into protein density and interaction with drugs by quantifying
relationships to yeast homologues: class I (HDACs 1, 2, 3,
the distribution of a radiotracer with extraordinarily high
and 8), class IIa (HDACs 4, 5, 7, and 9), class IIb (HDACs 6,
10), class III (Sirtuins 1−7), and class IV (HDAC 11).
Abnormal expression levels (both increased and decreased
expression) of HDAC 1, 2, 3, and 6 that have been correlated
with many diseases, including several forms of cancer, heart
failure, inflammatory diseases, and cognitive and psychiatric
sensitivity. To date there are no validated tools for assessing the
densities of HDAC enzymes in human despite the importance
of these enzymes and their remarkably high expression. Efforts
by others have resolved non-invasive probes for imaging
HDAC enzyme density in vivo,19−23 however these tools have
disorders (Table 1). However, our knowledge of normal
HDAC density and alterations in HDACs with human disease
remains extremely limited and invasive methodologies used so
Received: June 9, 2014
© XXXX American Chemical Society
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Table 1. Selected Evidence of HDAC 1, 2, 3, and 6
Involvement in Diseases
selected evidence of HDAC 1, 2, 3, and 6
disease/process
involvement
refs
1, 2
HDAC 1 and 3 protein complex integrity links
deficient DNA repair with neurotoxicity
HDAC 1 expression is elevated in vulnerable brain
regions of mouse disease models
3
4
neurodegenerative
disorders
HDAC 2 is elevated in Alzheimer’s patient brain and
negatively impacts memory in animal models
HDAC 6 overexpression in patients with AD
5
6
HDAC 1 overexpression in the prefrontal cortex
disrupts working memory
learning and memory
heart failure
HDAC 2 loss improves working memory and
accelerates extinction learning
7
8
HDAC 1 and 2 are critical mediators of autophagy
and cardiac plasticity
Class I HDAC enzymes alter cardiomyocyte
hypertrophy via ERK kinase activity
9
asthma
HDAC 2 deficiency in cells blunts transcriptional
response to steroids and anti-inflammatory drugs.
10
11
12
cancer (general)
breast cancer
HDAC acetylation of p53 mediates tumor cell
survival
HDAC 6 mRNA expression may have potential
both as a marker of endocrine responsiveness and
as a prognostic indicator in breast cancer
Figure 1. [11C]6: a translational PET imaging probe. We have
developed a potent HDAC imaging agent, termed [11C]6,
incorporating three key structural features to create a versatile and
translational probe for visualizing HDAC expression in vivo.
Intravenous injection of trace amounts of [11C]6 (nanogram scale)
in baboon and imaging by PET-MR demonstrates quantifiable uptake
in the brain and in diverse peripheral organs. This illustrates the
potential for [11C]6 as a broadly applicable tool in evaluating HDAC
density in humans.
HDAC 3 turnover is induced in cells by
isothiocyanate therapeutics
13
14
colon cancer
HDAC 1 and NuRD complex contributes to
epigenetic silencing of colorectal tumor
suppressor genes
HDACs 1−3 overexpression confer cisplatin
resistance in ovarian cancer cell lines
15
ovarian cancer
HDAC 1 and 3 overexpression mediate proliferation 16
and migration of ovarian cancer cells
RESULTS
■
Design and Synthesis of PET Imaging Agents with
Brain Permeability. HDAC inhibitors comprise diverse
structural classes including hydroxamic acids, natural cyclic
peptides, and short-chain fatty acids as well as aminoanilines
and ketones. Most classes of HDAC inhibitors contain three
main structural elements to their pharmacophore: (i) a capping
group, (ii) a linker domain, and (iii) a zinc-binding group,
critical for enzyme action. Modification of these structural
elements has so far revealed that the brain uptake of most
HDAC inhibitors is limited.19,24 To develop an HDAC PET
probe with broad disease applicability, we wanted to integrate a
chemical structure that would exhibit robust exposure to both
peripheral organs and the brain. In medicinal chemistry efforts
by others, the adamantyl group has been identified as a
structural subunit that confers CNS distribution in several
drugs classes including antiviral drugs,25 cannabinoid receptor
modulators.26 Further, adamantane-based hydroxamic acids
were recently reported and showed strong evidence of HDAC
inhibition.27 Using these previous studies to guide our synthesis
efforts, we designed compound 6, a cinnamic acid-based
HDAC inhibitor containing an adamantyl group as a ‘blood
brain barrier (BBB) carrier’ (Figure 1). Compound 6 was
synthesized through reductive amination, followed by con-
version into a hydroxamic acid in the presence of hydroxyl-
amine and sodium hydroxide. The radiosynthesis of [11C]6 was
accomplished using its desmethyl precursor in DMSO with
[11C]methyl iodide ([11C]CH3I) as outlined in Scheme 1.
Physicochemical Properties of 6 and ex Vivo Binding.
The in vitro inhibitory activities of 6 were measured for each
HDAC isoform HDAC1 through HDAC9 (Table 2) using
recombinant human enzymes, with IC50 values for the FDA
rapid binding kinetics and poor brain penetrating ability or bind
to a different subset of HDACs than the isoforms that have
been extensively implicated in diseases via study of patients and
preclinical animal models.
Herein, we report the development of [11C](E)-3-(4-
((((3r,5r,7r)-Adamantan-1-ylmethyl)(methyl)amino)methyl)-
phenyl)-N-hydroxyacrylamide ([11C]Martinostat, [11C]6) (Fig-
ure 1) as a molecule ready for translation to human PET
imaging (HDAC 1, 2, 3, and 6). Compound 6 is an
adamantane-based hydroxamic acid with selective binding to
HDAC 1, 2, 3, and 6 with subnanomolar potency and fast
binding kinetics. Using rodent and nonhuman primate PET
imaging, we demonstrate robust uptake and high specific
binding in brain, heart, spleen, kidney, and pancreas. Using this
tool, measurement of HDAC density of target isoforms 1, 2, 3,
and 6 is possible in peripheral organs and brain for the first
time. Measuring HDAC target density/occupancy with the
aformentioned non-invasive tool promises a major advance to
understand epigenetic mechanism in normal body tissues and
diseases. Self-blocking experiments have demonstrated the
sensitivity of [11C]6 to resolve differences in HDAC expression
in peripheral organs and brain. Further, blocking experiments
with the clinical approved HDAC inhibitor, SAHA, underscore
the utility of this tool to evaluate HDAC density and target
engagement with different therapeutics. Taken together, our
findings highlight the immediate utility of [11C]6 as a
preclinical PET tool to investigate HDAC expression in animal
models. Furthermore, we outline and discuss the optimal
properties of [11C]6 as an HDAC radiotracer that strongly
support its development as a clinical research tool.
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a
Scheme 1. Synthesis of 6, Its Radiolabeling Precursor (4) and [11C]6
a
Reagents and conditions: (a) NaBH4, MeOH, overnight, rt, 75%; (b) formaldehyde, AcOH, NaBH4, MeOH, rt, overnight, 55%. (c) NH2OH (aq),
1M NaOH, MeOH/THF, 0 °C to rt, 4 h, 42% for 6, 40% for 5; RCY of [11C]6, 3−5% (non-decay corrected to trapped [11C]CH3I).
To test the selective binding of 6 over other potential protein
targets, we screened the percentage inhibition of 6 (50 nM)
using radioligand displacement assay against 4 zinc-dependent
enzymes including matrix metalloproteinases and 80 additional
non-HDAC or Zn-dependent central nervous system (CNS)
targets. We determined minimal if any off-target binding,
indicating high selectivity of 6 to the HDAC family (Table S1).
The only potential off-target binding was the dopamine
transporter (DAT) as implicated by the in vitro test. 6 (50
nM) resulted in a 24% inhibition of DAT (where 50%
inhibition is the minimum threshold to identify a potential
binding event). Therefore, to exclude a putative binding
interaction of 6 to DAT (given that it is a relatively high
dentistry brain protein), we used the established radiotracer
[11C]β-CFT to evaluate DAT density/occupancy in rats and
observed no impact of 6 treatment (1 mg/kg, ip, 90 min prior)
(Figure S2).
Table 2. HDAC Selectivity, Potency, and Efficacy of 6
Compared To SAHA
a
assay
6
SAHA
IC50 (nM)
0.3
HDAC 1
HDAC 2
HDAC 3
HDAC 4
HDAC 5
HDAC 6
HDAC 7
HDAC 8
HDAC 9
4.0
11
2.0
0.6
3.0
1970
>30000
8750
2.0
352
4.1
>20000
>15000
>15000
EC50 (nM)
100
>30000
1020
>30000
H3K9ac
3400
1900
H4K12ac
100
a
In vitro IC50 (nM) values using recombinant human enzymes for
HDAC subtypes 1−9 and specific substrates demonstrate that 6 is a
selective inhibitor of HDAC 1−3 (0.3−2.0 nM) with decreased
potency to inhibit HDAC 6 (4.1 nM) or other subtypes (>352 nM).
Comparatively, the hydroxamate HDAC inhibitor SAHA exhibited
lower affinity for HDAC targets 1−3 (3.0−11 nM). Dose−response
plots in cultured primary mouse neuronal cells measuring relative
H3K9ac and H4K12ac levels revealed potent induction of histone
acetylation (EC50) by 6 (100 nM) compared to SAHA (1900−3400
nM).
To further establish that [11C]6 binding was specific for
HDAC targets, we conducted blocking studies via auto-
radiography on rat brain sections ex vivo. We observed that
specific binding of [11C]6 was reduced by preincubation of
sagittal rat brain sections with either of the unlabeled HDAC
inhibitors: SAHA, a hydroxamic acid-based HDAC inhibitor
(selective for class I and IIb HDAC isoforms), or CI-994, a
benzamide-based HDAC inhibitor (selective for HDAC 1, 2,
and 3 isoforms) (Figure S1C,D).
In Vivo PET-CT Imaging with [11C]6 in Rodents. High
Brain Uptake and Reversible Binding. To first test [11C]6 as a
radiotracer in vivo, we conducted PET imaging focused on rat
brain. Using PET-CT, we determined that [11C]6 exhibited
high BBB penetration and sustained binding over the scanning
time (60 min) when administered by intravenous bolus
injection (0.9−1.1 mCi per animal), as shown in Figure S3A.
To confirm that [11C]6 was not effluxed from the brain by the
xenobiotic pump, P-glycoprotein (P-gp), we measured radio-
ligand uptake in brain after treatment with the P-gp inhibitor,
cyclosporin A (CsA), and found no difference compared to
control treatment (Figure S4A). Additionally, [11C]6 binding in
the rat brain was disrupted by an iv challenge with unlabeled 6
20 min after tracer administration as evidenced by a rapid
decrease in the time−activity curve slope. This rapid loss of
approved HDAC inhibitor drug, suberoylanilide hydroxamic
acid, vorinostat (SAHA), measured in parallel as a reference (to
assess the variability among different assays). Compound 6
showed nanomolar IC50 values for class-I HDAC isoforms
(HDAC 1−3, 0.3, 2.0, and 0.6 nM, respectively), and the class-
IIb HDAC 6 (4.1 nM), with >100-fold selectivity over each of
the other HDAC isoforms assayed. Using [11C]6 in vitro, we
measured the partition coefficient (log D) at 2.03 and
calculated tPSA of 52.5, which are in the preferred range for
brain penetration, and further detected plasma protein binding
(PPB), as measured in blood from baboon (1.4% unbound)
and human (5.5% unbound), at levels consistent with those for
the clinical dopamine D2 receptor radiotracer, [11C]raclopride28
(Figure S1).
C
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Figure 2. Kinetic modeling results with [11C]6 in baboon brain. (A) The total volume of distribution (VT) images from one representative animal
show robust differences in radiotracer uptake at baseline and after blocking (0.5 mg/kg iv, 10 min pretreatment); (B) Two independent baseline-
blocking studies were used to resolve quantitative VT data which show that pretreatment with unlabeled 6 (0.5 or 1.0 mg/kg) dose-dependently
blocks tracer uptake in different baboon brain regions. WB: whole brain; CB: cerebellum; M1: primary motor cortex ; PU: putamen; TH: thalamus;
V1: primary visual cortex ; CA: caudate; WM: white matter.
Figure 3. [11C]6 PET-MR imaging. Axial views of summed PET images (40−80 min) superimposed with MR images from the same baboon
following injection of radiotracer (4 mCi/baboon). Images illustrate tracer uptake in organs of interest at baseline and after pretreatment with
unlabeled 6 (0.5 mg/kg). Robust blocking was observed in organs of interest including: A, heart; B, spleen; C, kidneys; and D, pancreas. Time−
activity curves (baseline, blue; blocking, red) demonstrate a high specific binding of [11C]6 in these peripheral organs as the percent injected tracer
dose per cm3 tissue is markedly reduced by blocking.
radioactivity from brain regions provided confirmation of the
reversibility of [11C]6 binding to CNS targets (Figure S4B).
To determine the stability of [11C]6 in brain, we measured
the presence of radiolabeled parent compound and polar
D
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metabolites in homogenized rat brain tissue 30 min post-in vivo
injection of [11C]6. By eluting extracted and homogenized rat
brain with solvents containing fractional increases of acetoni-
trile in water, we resolved a pattern of radioactivity indicating
intact parent compound in the brain with limited degradation
over the duration of a typical 30−90 min PET scan (Figure
S5A). A similar pattern indicating unmetabolized [11C]6 was
observed in baboon plasma, 5 min post-injection of the tracer
(Figure S5B).
Dose-Dependent Blockade. To investigate the specificity of
[11C]6, we performed PET imaging studies in rats with a 5 min
pretreatment of unlabeled 6 at different doses (0.001, 0.1, 0.5,
and 2.0 mg/kg). We found that [11C]6 binding in brain was
blocked in a dose-dependent manner with a stepwise reduction
in the percent tracer uptake after administration of unlabeled 6
(Figure S3B). To test the sensitivity of [11C]6 against other
HDAC inhibitors, we pretreated a rat with CN54, a highly brain
penetrant hydroxamic acid HDAC inhibitor as determined by
LC-MS-MS (brain/plasma = 4.5 after 20 min administration of
CN54 at 10 μg/kg via iv), and SAHA, a hydroxamic acid
HDAC inhibitor with limited brain penetrating ability.24 As
expected, there was robust in vivo blockade of radioactivity in
brain with the treatment of CN54, but not SAHA (Figure
S3C).
Therefore, 50−80% of the signal represented specific binding
in the selected brain regions suggesting that [11C]6 has
sufficient sensitivity to assess a wide range of CNS diseases in
which HDAC may be altered, such as Alzheimer’s disease and
in mood disorders,4,31,32 and for evaluation of drug-HDAC
engagement.
High uptake and blockade in peripheral organs. We
further evaluated the tracer uptake and blockade in peripheral
organs in baboons. [11C]6 showed high uptake in major organs
including heart, kidney, pancreas, and spleen (Figure 3). The
uptake in these organs was markedly decreased by pretreatment
with unlabeled 6, indicating that the [11C]6 signal also
represents HDAC expression in peripheral organs. To further
confirm the sensitivity of [11C]6 to detect changes in HDAC
occupancy, we measured radiotracer uptake in the baboon both
before and after 75 min infusion of SAHA (5 mg/kg, iv). We
found that radiotracer uptake in peripheral tissue was robustly
blocked by SAHA infusion, consistent with efficient distribution
of this HDAC inhibitor blocking agent in nonbrain organs24
(Figure S6).
DISCUSSION
■
Epigenetic therapies that target regulatory mechanisms of the
human genome represent an emerging class of novel
therapeutic agents. The importance of understanding aberrant
enzyme expression of chromatin modifying enzymes including
HDACs is highlighted by the discovery that protein coding
mutations may play a role in human disease pathologies linked
to cancer in major organs, cardiac dysfunction, and CNS
disorders. The impact of these enzyme density changes is
further implicated in disease as recent large-scale genome-wide
association studies (GWAS) have shown that gene regulatory
regions key in many common human diseases harbor an
enrichment of single nucleotide polymorphisms (SNPs).33−38
This evidence of deregulated gene expression mechanism
points to a more general need to target epigenetic machinery
that may be causally involved in disease pathogenesis.
Therefore, developing therapeutics for many human diseases
can be best accomplished by first understanding the density and
distribution of epigenetic machinery including HDAC enzymes
in vivo.
In Vivo PET-MR Imaging with [11C]6 in Baboon. High
Uptake in the Brain and Dose-Dependent Blockade. Next,
we chose to utilize nonhuman primates to evaluate the
distribution and specificity of [11C]6. In baboons, we found
that high brain uptake (50−60 in VT, total volume of
distribution) of [11C]6 based on PET-MR focused on the
head. Similar to the blocking results we obtained in rats,
injection of unlabeled 6 (0.5 and 1.0 mg/kg, iv) dose-
dependently reduced the radioactivity uptake (measured as
VT) in specific brain regions including cerebellum, cortex,
putamen, and caudate (Figure 2). The averaged Bmax of whole
brain, 19.6 2.9 μM of protein, was calculated from the PET
image on the basis of the established relationship between total
binding and dissociation rate29 and was consistent with ex vivo
protein quantification in rodents by two methods.30 Physio-
logical effects (i.e., respiration rate, blood pressure, heart rate,
and end-tidal CO2) were unchanged at any administered dose
of 6 or [11C]6. The baboons used in these imaging studies were
not sacrificed.
Development of specific PET radiotracers to measure HDAC
density or any other epigenetic component, with high uptake
and major organs, is challenging.39 There are a number of
major factors (summarized here A through D) that determine
the success of a radiotracer candidate. Some factors can be
predicted from in vitro assays (e.g., binding affinity and
selectivity), and those which are more empirically determined
(e.g., specific to nonspecific binding ratio). (A) The binding
affinity and selectivity of a radiotracer for its target must be high
enough to produce sufficient signal to background for detection
and selective for the target so that images represent the
biological target. (B) Additionally, radiotracers must exhibit
specific binding, which is challenging to predict but can be
easily measured by presaturating the target with an unlabeled
(nonradioactive) form of the radiotracer thus resulting in
decreased binding of radiotracer to its target. Unlabeled
compound binding is also expected to alter the distribution
and pharmacokinetics of the radioligand. (C) For CNS
imaging, small molecules must cross the BBB. In general,
small molecular weight (<400 Da) molecules with relatively
high lipophilicity are required for a tracer to sufficiently
penetrate the BBB. Predictive models based on PET imaging
Arterial Plasma Analysis. After [11C]6 intravenous bolus
injection, radioactivity in arterial plasma clears rapidly as
expected and as depicted in Figure S5C. Polar metabolites were
observed in the plasma (50% of the plasma radioactivity after
10 min); however, radioactivity in brain tissue of rodents at late
time points (30 min post administration) was associated almost
exclusively with the parent molecule. These data support that
the PET images for the brain tissue largely represent [11C]6
and not radiolabeled metabolites. For quantitative analysis of
the NHP PET imaging data, a metabolite-corrected arterial
input function was used.
Kinetic Modeling Shows High Specific Binding. Two-tissue
compartmental modeling was applied to analyze dynamic [11C]
6 PET data, corrected for arterial input function and parent
compound metabolism to interpret results from intact and
perfused radiotracer. Based on the modeling results (Figure 2),
blocking with 0.5 and 1.0 mg/kg, VT decreased in all brain
regions. Based on our rodent experiments and dose-
equilibration for body mass, we assumed that a 1.0 mg/kg
dose would provide full saturation of HDAC binding.
E
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synthesized (from Sai Life Sciences Ltd.) with >98% purity, as
determined by a high-pressure liquid chromatography (HPLC) system
(YMC TRIART C-18 (150 mm × 4.6 mm × 3 u) mobile phase: A:
0.05% TFA in water/B: 0.05% TFA in acetonitrile. Flow rate: 1.0 mL/
min. Gradient: 15% B to 95% B in 8.0 min, hold until 9.5 min, 15% B
in 13.0 min hold until 15.0 min. The quantitative NMR test were
conducted by Sai Life Sciences Ltd., and 1,4-dimethoxybenzene was
used as the reference. [11C]CO2 (1.2 Ci) was obtained via the 14N (p,
α)11C reaction on nitrogen with 2.5% oxygen, with 11 MeV protons
(Siemens Eclipse cyclotron), and trapped on molecular sieves in a
TRACERlab FX-MeI synthesizer (General Electric). [11C]CH4 was
obtained by the reduction of [11C]CO2 in the presence of Ni/
hydrogen at 350 °C and recirculated through an oven containing I2 to
produce 11CH3I via a radical reaction.
All animal studies were carried out at Massachusetts General
Hospital (PHS Assurance of Compliance no. A3596-01). The
Subcommittee on Research Animal Care (SRAC) serves as the
Institutional Animal Care and Use Committee (IACUC) for the
Massachusetts General Hospital (MGH). SRAC reviewed and
approved all procedures detailed in this paper.
PET-MR imaging was performed in anesthetized (ketamine,
isoflurane) baboon (Papio anubis) to minimize discomfort. Highly
trained animal technicians monitored animal safety throughout all
procedures, and veterinary staff were responsible for daily care. All
animals were socially housed in cages appropriate for the physical and
behavioral health of the individual animal. Animals were fed thrice per
diem, with additional nutritional supplements provided as prescribed
by the attending veterinarian. Audio, video, and tactile enrichment was
provided on a daily basis to promote psychological well-being. No
nonhuman primates were euthanized to accomplish the research
presented.
PET-CT imaging was performed in anesthetized (isoflurane) rats
(Sprague−Dawley) to minimize discomfort. Highly trained animal
technicians monitored animal safety throughout all procedures, and
veterinary staff were responsible for daily care. All animals were socially
housed in cages appropriate for the physical and behavioral health of
the individual animal. Animals were given unlimited access to food and
water, with additional nutritional supplements provided as prescribed
by the attending veterinary staff. Animals were euthanized at the end
of the study using sodium pentobarbital (200 mg/kg, ip).
Chemical Synthesis. (E)-Methyl 3-(4-((((3r,5r,7r)-adamantan-1-
ylmethyl)amino)methyl)phenyl)acrylate (3). Adamantan-1-ylmethan-
amine (1) (1g, 6.0 mmol) and (E)-methyl 3-(4-formylphenyl)acrylate
(2) (1 g, 5.3 mmol) was dissolved in MeOH (30 mL), and the mixture
was stirred at room temperature for 2 h. Sodium borohydride (0.61g,
16 mmol) was then added, and the suspension was stirred overnight at
room temperature. The white precipitate was filtered and dried to
obtain the product 3 (1.35 g, yield: 75%).1H NMR (500 MHz,
CDCl3): δ 7.69 (d, J = 16 Hz, 1H), 7.48 (d, J = 7 Hz, 2H), 7.35 (d, J =
7 Hz, 2H), 6.43 (d, J = 16 Hz, 1H), 3.81 (s, 2H), 3.80 (s, 3H), 2.23 (s,
3H), 1.96 (s, 3H), 1.63−1.73 (m, 6H), 1.53 (s, 6H); 13C NMR (125
MHz, CDCl3): 167.55, 144.78, 143.81, 132.87, 128.35 (2C), 128.08
(2C), 117.10, 62.15, 54.28, 51.65, 40.85 (3C), 37.24 (3C), 33.49,
28.48 (3C). LC-MS calculated for C22H29NO2 expected [M]: 339.2;
Found [M + H]+: 340.3.
(E)-3-(4-((((3r,5r,7r)-Adamantan-1-ylmethyl)amino)methyl)-
phenyl)-N-hydroxyacrylamide (4). To a solution of 3 (0.5 g, 1.5
mmol) in MeOH/THF (5 mL/5 mL) at 0 °C was added NH2OH
(50% aq solution, 3 mL) followed by 1 M NaOH (2 mL). The mixture
was stirred at 0 °C for 2 h, warmed to room temperature, and stirred
for 2 h. Acidification with 1 M HCl to pH 7−8 (pH paper) resulted in
product precipitation. The precipitate was filtered and dried to obtain
4 (0.2 g, 40%) as white solid. 1H NMR (500 MHz, CDCl3): δ 7.69 (d,
J = 16 Hz, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.39 (d, J = 7.5 Hz, 2H), 6.42
(d, J = 16 Hz, 1H), 3.80 (s, 3H), 3.54 (s, 2H), 2.19 (s, 3H), 2.10 (s,
2H), 1.95 (s, 3H), 1.62−1.723 (m, 6H), 1.53 (s, 6H); 13C NMR (125
MHz, CDCl3): 164.92, 141.31, 139.69, 133.75, 128.61 (2C), 127.42
(2C), 116.88, 71.09, 61.08, 53.31, 40.41 (3C), 36.77 (3C), 32.84,
28.48 (3C). LC-MS calculated for C21H28N2O2 expected [M]: 340.5;
data are emerging for HDAC inhibitors that suggest the total
polar surface area (tPSA) needs to be <65.22 (D) With all of the
above properties satisfied, a tracer candidate can still be
rendered unusable in vivo if it is metabolized rapidly and those
metabolites pervade the tissue regions of interest. Each of the
primary factors for a successful radiotracer is met by [11C]6 and
supported by our biochemical and preclinical imaging data. Of
the more than 100 compounds we assessed through our tracer
identification work, [11C]6 was unique. While we have observed
some trends in molecular properties, structure, and in vivo
behavior, fully understanding the molecular basis for [11C]6
success will require additional structure−activity relationship
determination and computational modeling.
Our data support [11C]6 as a new tool to visualize HDAC
density in the body and brain. The robust and blockable brain
uptake with [11C]6 as a PET probe can be used to quantify
HDAC density in brain disorders, such as neurodegenerative
diseases and mood disorders, and for demonstrating target
engagement of putative HDAC inhibitor therapeutics early in
compound development pipeline. We found stable probe
uptake in diverse peripheral organs in nonhuman primates,
each with a distinct profile of HDAC target density and affinity
as evidenced by time−activity curve (TAC) shape (Figure S6).
In each case, we further demonstrated that [11C]6 binding
could be prevented using a known, clinically applied HDAC
inhibitor drug (Figure S6) illustrating the direct utility of this
probe for understanding systemic impact of a cancer
therapeutic. A recent report demonstrates that class I HDAC
inhibition by a prototypical HDACi therapeutic, MS-275,
stimulates renal dysfunction.40 Dose-limiting toxicity may not
be surprising given the widespread expression and central role
of HDACs in normal biological function. However, this
underscores a mechanism-based liability for HDAC inhibitor
leads proposed to date as CNS disease therapeutics where large
mass dosing strategies are used to reach effective concentrations
of HDACi in brain.24,41,42 Importantly, the ability to
simultaneously measure HDAC target engagement in periph-
eral organs and brain is a major development in not only
understanding CNS disease mechanisms but also in prioritizing
novel HDAC inhibitors with minimal systemic burden.
In summary, [11C]6 binds to HDAC subtypes (HDAC 1, 2,
3, and 6) with high selectivity and specificity and provides a
tool for quantitative imaging of HDAC density in the brain and
in peripheral organs in vivo. [11C]6 can be used as a PET
radioligand for studying HDACs in disorders where altered
regulation of HDACs is found. The approach is also valuable
for evaluation of potential drugs in vivo, therapy response can
be compared in the same animal before drug treatment. Safety
and toxicology studies have been completed, and we are
progressing [11C]6 forward for evaluation as the first HDAC
PET radiotracer and a novel class of epigenetic imaging probes
in humans.
EXPERIMENTAL SECTION
■
General Methods and Materials. All reagents and solvents were
of ACS-grade purity or higher and used without further purification.
NMR data were recorded on a Varian 500 MHz magnet and were
reported in ppm units downfield from trimethylsilane. Analytical
separation was conducted on an Agilent 1100 series HPLC fitted with
a diode-array detector, quaternary pump, vacuum degasser, and
autosampler. Mass spectrometry data were recorded on an Agilent
6310 ion trap mass spectrometer (ESI source) connected to an Agilent
1200 series HPLC with quaternary pump, vacuum degasser, diode-
array detector, and autosampler. The precursor and compound 6 were
F
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Found [M + H]+: 341.3. HPLC purity: 98.09%. Quantitative NMR
purity: 95.10%.
and then fixed in 4% formaldehyde for 10 min. Following two washes
with phosphate-buffered saline, cells were permeabilized and blocked
with 0.1% Triton X-100 and 2% BSA in PBS. Cells were stained with
an anti-Ac-H4K12 antibody (Millipore, catalog no. 04−119) and
Alexa488-conjugated secondary antibody (Molecular Probes). Cell
nuclei were identified by staining with Hoechst 33342 (Invitrogen,
H3570). Cell nuclei and histone acetylation signal intensity were
detected and measured using a laser-scanning microcytometer
(Acumen eX3, TTP Laptech). Acumen Explorer software was used
to identify a threshold of histone acetylation signal intensity such that,
without HDAC inhibitor, >99.5% of cells had intensity levels below
the threshold. In the presence of HDAC inhibitors, cells signal
intensities above the threshold were scored as “bright green cells” and
expressed as a percentage normalized to DMSO controls. EC50 values
were determined from curve fitting using GraphPad Prism v5 software
(GraphPad Software, Inc. La Jolla, CA, USA).
CNS Target Binding Assay. 6 was submitted to a panel of 80
binding assays for common transmembrane and soluble receptors, ion
channels, and monoamine transporters in the CNS (High-Throughput
Profile P-3, Cerep, France). 6 was assayed in duplicate at 50 nM. This
concentration was >10× the IC50 values determined for HDAC targets
(0.3−4.1 nM, see Table 2, main text) and in vast excess of the amount
of 6 administered in PET imaging experiments. For example, injected
dose = 1 mCi/rat; specific activity (see Figure S2) = 1 mCi/1 nmole;
therefore, calculated dose = 1 nmole 6/ rat. Inhibition of control
binding did not exceed 45% for any target assayed, supporting that 6
predominantly binds HDAC targets.
(E)-Methyl 3-(4-((((3r,5r,7r)-adamantan-1-ylmethyl) (methyl)
amino) methyl) phenyl) acrylate (5). To a solution of 3 (0.5 g, 1.5
mmol) in MeOH (30 mL) was added formaldehyde (33% aq solution,
2 mL) followed by acetic acid (0.1 mL). The mixture was stirred at
room temperature for 2 h. Sodium borohydride (0.61g, 16 mmol) was
then added, and the suspension was stirred overnight at room
temperature. The white precipitate was filtered and purified by flash
chromatography in hexanes:ethyl acetate (4:1) to obtain the product 3
(0.29 g, yield: 55%) as white solid. 1H NMR (500 MHz, MeOH-d4): δ
7.55 (d, J = 16 Hz, 1H), 7.52 (d, J = 7.5 Hz, 2H), 7.37 (d, J = 7.5 Hz,
2H), 6.47 (d, J = 16 Hz, 1H), 3.77 (s, 2H), 2.21 (s, 2H), 1.94 (s, 3H),
1.66−1.76 (m, 6H), 1.54−1.55 (m, 6H); 13C NMR (125 MHz,
MeOH-d4): 164.93, 141.32, 139.71, 133.76, 128.62 (2C), 127.43 (2C),
116.8901, 61.08, 53.32, 40.42 (3C), 36.78 (3C), 32.84, 28.48 (3C).
LC-MS calculated for C21H28N2O2 expected [M]: 353.2; Found [M +
H]+: 354.1.
(E)-3-(4-((((3r,5r,7r)-Adamantan-1-ylmethyl)(methyl)amino)-
methyl)phenyl)-N-hydroxyacrylamide (6). To a solution of 5 (0.5 g,
1.4 mmol) in MeOH/THF (3 mL/3 mL) at 0 °C was added NH2OH
(50% aq solution, 3 mL) followed by 1 M NaOH (2 mL). The mixture
was stirred at 0 °C for 2 h, warmed to room temperature, and stirred
for 2 h. Acidification with 1 M HCl to pH 7−8 (pH paper) resulted in
product precipitation. The precipitate was filtered and dried to obtain
1
6 (0.21 g, 42%) as white solid. H NMR (500 MHz, DMSO-d6): δ
7.49 (d, J = 7.5 Hz, 2H), 7.42 (d, J = 16 Hz, 1H), 7.33 (d, J = 7.5 Hz,
2H), 6.45 (d, J = 16 Hz, 1H), 3.48 (s, 2H), 2.11 (s, 3H), 2.06 (s, 2H),
1.89 (s, 3H), 1.55−1.65 (m, 6H), 1.46 (s, 6H); δ 13C NMR (125
MHz, DMSO-d6): 163.15, 142.00, 138.43, 139.91, 129.36 (2C),
127.77 (2C), 119.06, 70.12, 64.57, 45.71, 41.01 (3C), 37.21 (3C),
35.27, 28.33 (3C). LC-MS calculated for C22H30N2O2 expected [M]:
354.2; Found [M + H]+: 355.3. HPLC purity: 99.64%. Quantitative
NMR purity: 97.05%.
Radiosynthesis of [11C]6. [11C]CH3I was trapped in a TRACERlab
FX-M synthesizer reactor (General Electric) preloaded with a solution
of precursor (4) (1.0 mg) in dry DMSO (300 μL). The solution was
stirred at 100 °C for 4 min, and water (1.2 mL) was added. The
reaction mixture was purified by reverse phase semipreparative HPLC
(Phenomenex Luna 5u C8(2), 250 × 10 mm, 5 μm, 5.0 mL/min, 40%
H2O + ammonium acetate (0.1 M)/ 60% CH3CN), and the desired
fraction was collected. The final product was reformulated by loading
onto a solid-phase exchange (SPE) C-18 cartridge, rinsing with 1 M
NaOH aq (5 mL), eluting with EtOH (1 mL), and diluting with 50 μL
acetic acid in saline (0.9%, 9 mL). The chemical and radiochemical
purity of the final product was tested by analytical HPLC (Agilent
Eclipse XDB-C18, 150 × 4.6 mm). The identity of the product was
confirmed by analytical HPLC with additional co-injection of reference
standard. The average time required for the synthesis from end of
cyclotron bombardment to end of synthesis was 35 min. The average
radiochemical yield was 3−5% (nondecay corrected to trapped
[11C]CH3I). Chemical and radiochemical purities were ≥95% with a
specific activity 1.0 0.2 Ci/μmol (EOB).
HDAC Inhibition Assay. All recombinant human HDACs were
purchased from BPS Bioscience. The substrates, Broad Substrates A
and B, were synthesized in house. All other reagents were purchased
from Sigma-Aldrich. Caliper EZ reader II system was used to collect all
data. HDAC inhibition assays: Compounds were tested in a 12-point
dose curve with 3-fold serial dilution starting from 33.33 μM. Purified
HDACs were incubated with 2 μM (the concentration is kept the
same for all the HDACs, below Km of substrate) carboxyfluorescein
(FAM)-labeled acetylated or trifluoroacetylated peptide substrate
(Broad Substrates A and B, respectively) and test compound for 60
min at room temperature, in HDAC assay buffer that contained 50
mM HEPES (pH 7.4), 100 mM KCl, 0.01% BSA, and 0.001% Tween-
20. Reactions were terminated by the addition of the known pan
HDAC inhibitor LBH -589 (panobinostat) with a final concentration
of 1.5 μM. Substrate and product were separated electrophoretically,
and fluorescence intensity in the substrate and product peaks were
determined and analyzed by Labchip EZ Reader. The reactions were
performed in duplicate for each sample. IC50 values were automatically
calculated by Origin8 using 4 Parameter Logistic Model. The percent
inhibition was plotted against the compound concentration, and the
IC50 value was determined from the logistic dose−response curve
fitting by Origin 8.0 software.
Specific Activity of [11C]6. Specific radioactivity (SA) of [11C]6 was
calculated from an analytical HPLC sample of 100 μL. A calibration
curve of known mass quantity versus HPLC peak area (254 nm) was
used to calculate the mass concentration of the 100 μL. SA was
determined using the mass concentration value determined for the
formulated [11C]6 solution, volume, and measured radioactivity. The
identity of the mass peak assigned to [11C]6 was supported by a
second HPLC injection, where [11C]6 was mixed with nonradioactive
compound 6 and yielded a single peak. The calibration curve used
appears in Figure S1.
Mouse Primary Neuronal Histone Acetylation Assays. Mouse
primary neuronal cultures were generated in factory precoated poly-D-
lysine 96-well plates (BD Biosciences no. BD356692) treated
overnight with 75 μL/well of laminin [0.05 mg/mL] (Sigma no.
L2020) in PBS buffer. E15 embryonic mouse forebrain was dissociated
into a single cell suspension by gentle trituration following trypsin/
DNase digestion (trypsin: Cellgro no. 25-052-Cl; DNase: Sigma no.
D4527). 12,500 cells per well were plated in 100 μL neurobasal
medium (Gibco no. 21103-049) containing 2% B27 (Gibco no. 17504-
044) and 1% penicillin/streptomycin/glutamine (Cellgro no. 30-009-
CI) and cultured at 37 °C with 5% CO2. After 13 days, cultures were
treated with HDAC inhibitors by pin transfer of compound (185 nL
per well) using a CyBi-Well vario pinning robot (CyBio Corp.,
Germany) and subsequently incubated for 24 h at 37 °C with 5% CO2
Log D Determination. An aliquot (∼50 μL) of the formulated
radiotracer was added to a test tube containing 2.5 mL of octanol and
2.5 mL of phosphate buffer solution (pH 7.4).The test tube was mixed
by vortex for 2 min and then centrifuged for 2 min to fully separate the
aqueous and organic phase. A sample taken from the octanol layer (0.1
mL) and the aqueous layer (1.0 mL) was saved for radioactivity
measurement. An additional aliquot of the octanol layer (2.0 mL) was
carefully transferred to a new test tube containing 0.5 mL of octanol
and 2.5 mL of phosphate buffer solution (pH 7.4). The previous
procedure (vortex mixing, centrifugation, sampling, and transfer to the
next test tube) was repeated until six sets of aliquot samples had been
prepared. The radioactivity of each sample was measured in a well
counter (PerkinElmer, Waltham, MA).The log D of each set of
samples was derived by the following equation: log D = log (decay-
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corrected radioactivity in octanol sample × 10/decay-corrected
radioactivity in phosphate buffer sample).
coil arrays for nonhuman primate brain imaging with a PET resolution
of 5 mm and field of view of 59.4 and 25.8 cm (transaxial and axial,
respectively). Dynamic PET image acquisition was initiated followed
by administration of the radiotracer in a homogeneous solution of 10%
ethanol and 90% isotonic saline. An MEMPRAGE sequence began
after 30 min of the baseline scan for anatomic co-registration. To
characterize the specific binding of [11C]6, a second imaging
experiment was carried out in which unlabeled 6 was co-administered
intravenously at the start of acquisition. Both scans were carried out in
the same animal on the same day, separated by 2.5 h. In both scans, 4−
5 mCi of [11C]6 was administered to the baboon. Dynamic data from
the PET scans were recorded in list mode and corrected for
attenuation. Baboon data were reconstructed using a 3D-OSEM
method resulting in a fwhm resolution of 4 mm.
Plasma Protein Binding Assay. An aliquot of radiotracer in saline
(10 μL) was added to a sample of baboon or human plasma (0.8 mL).
The mixture was gently mixed by repeated inversion and incubated for
10 min at room temperature. Following incubation a small sample (20
μL) was removed to determine the total radioactivity in the plasma
sample (AT; AT = Abound + Aunbound). An additional 0.2 mL of the
plasma sample was placed in the upper compartment of a Centrifree
tube (Amicon, Inc., Beverly, MA) and then centrifuged for 10 min.
The upper part of the Centrifree tube was discarded, and an aliquot
(20 μL) from the bottom part of the tube was removed to determine
the amount of radioactivity that passed through the membrane
(Aunbound). Plasma protein binding was derived by the following
equation: %unbound = Aunbound × 100/AT.
Body imaging. Simultaneous PET-MR data were acquired using a
Siemens Biograph mMR system (Siemens Medical Solutions U.S.A.,
Inc., Malvern, PA). MR body imaging was performed with real-time
respiratory bellows-gating and using the Body Matrix coil and the
built-in spine coil as the receiving coil elements. High-resolution
anatomical T1W axial, coronal, and sagittal dual-echo scan with the
following parameters: TR 115 ms, TE1 1.23 ms, TE2 2.46 ms, matrix
size 256 × 168, FOV 35 cm, phase FOV 65.6%) and 4 mm slice
thickness were obtained. Additionally, in particular for the purpose of
optimal visualization of the pancreas, high-resolution fat-suppressed
axial T2W turbo spin echo (TSE) data were obtained with the
following parameters: TR 5264 ms, TE 98 ms, matrix size 488 × 235,
FOV 18.0 cm, phase FOV 75%, FA 150°, and 3 mm slice thickness.
For the purpose of MR-based attenuation correction (MRAC) of the
PET data, default T1-weighted (T1W) 2-point Dixon 3D volumetric
interpolated breath-hold examination (VIBE) scans were obtained.
PET data were obtained using a single-bed acquisition with an axial
field of view of 25.8 cm, transaxial field of view of 59.4 cm, and axial
and transaxial resolution of ≤4.8 and ≤4.7 mm, respectively. PET data
were acquired dynamically for 80 min after administration of the
radiotracer in a homogeneous solution of 10% ethanol and 90%
isotonic saline. To characterize the specific binding of [11C]6, a second
imaging experiment was carried out in which unlabeled 6 was co-
administered intravenously at the start of acquisition. Both scans were
carried out in the same animal on the same day, separated by 2.5 h. In
both scans, 4−5 mCi of [11C]6 was administered to the baboon. In
order to evaluate the blocking of [11C]6 by SAHA, an separate dual-
injection study of [11C]6 was performed. In that experiment
continuous SAHA infusion (2 mg/mL at 0.16 mL/min) was started
after 45 min of baseline scan with [11C]6, and the infusion was
continued during the 80 min PET scan after a second injection of
[11C]6. In all cases PET data were recorded in list mode, and
reconstruction was performed using a 3D-OSEM method resulting in a
fwhm resolution of 4 mm. Attenuation correction was performed using
the aforementioned 2-point Dixon 3D VIBE MR-derived attenuation
maps. Data of each 80 min scan were reconstructed for TAC analysis
using the following framing: 12 × 10, 3 × 20, 4 × 30, and 15 × 300 s.
For the dedicated self-blocking and SAHA blocking experiments,
additional static PET data were reconstructed using data obtained
between 40 and 80 min and 20−45 min, respectively, in order to
visually display the effects of blocking in multiple organs.
Ex vivo Autoradiography. Twenty μm thick sagittal rat brain
sections were cut using a −20 °C cryostat, thaw-mounted onto gelatin-
coated slides, fixed in 4% paraformaldehyde containing 2% ethanol for
60 min at 4 °C, and washed 10 min in ice-cold 10 mM Tris-HCl, pH
7.4. Sections were then incubated at room temperature in a 50 mL
bath containing either (i) 5% DMSO as control; (ii) unlabeled 6 (100
μM) or (iii) SAHA (100 μM) for 10 min; or (iv) CI-994 (100 μM) for
2 h to allow for slow HDAC binding kinetics known for benzamide
compounds including CI-994. All incubation baths contained 10 mM
Tris-HCl + 5% DMSO, pH 7.4. [11C]6 was prepared as described, and
100 μCi was added to each bath. Following 10 min incubation at room
temperature, sections were washed 2 × 1 min in 10 mM Tris-HCl (pH
7.4) and carefully wiped dry on absorbent towels. Sections were
exposed for 1 h to a multisensitive phosphorscreen and developed
using a Cyclone Plus phosphorimager (both from PerkinElmer).
Resulting parent image was evaluated using ImageJ software (NIH)
with JET color lookup table (LUT) with whole-image intensity
adjusted to enrich red color in control sections. Individual images of
sections were cropped using ImageJ with no additional adjustment to
color levels/thresholds.
Rodent PET-CT Acquisition and Post-Processing. Male Sprague−
Dawley rats were utilized in pairs, anesthetized with inhalational
isoflurane (Forane) at 3% in a carrier of 1.5−2 L/min medical oxygen,
and maintained at 2% isoflurane for the duration of the scan. The rats
were arranged head-to-head in a Triumph Trimodality PET/CT/
SPECT scanner (Gamma Medica, Northridge, CA). Rats were injected
standard references or vehicle via a lateral tail vein catheterization at
the start of PET acquisition. Dynamic PET acquisition lasted for 60
min and was followed by computed tomography (CT) for anatomic
coregistration. PET data were reconstructed using a 3D-MLEM
method resulting in a full width at half-maximum (fwhm) resolution of
1 mm. Reconstructed images were exported from the scanner in
DICOM format along with an anatomic CT for rodent studies. These
files were imported to PMOD (PMOD Technologies, Ltd.) and
manually co-registered using six degrees of freedom.
Rodent PET-CT Image Analysis. Volumes of interest (VOIs) were
drawn manually as spheres in brain regions guided by high-resolution
CT structural images and summed PET data, with a radius no <1 mm
to minimize partial volume effects. TACs were exported in terms of
decay corrected activity per unit volume at specified time points with
gradually increasing intervals. The TACs were expressed as percent
injected dose per unit volume for analysis.
Baboon PET-MR Acquisition. Papio anubis baboons, deprived of
food for 12 h prior to the study, were included in the PET-MR scans.
Atropine 0.05 mg/kg was used intramuscularly to prevent excessive
secretion (15 or 30 min of ketamine and Xylazine). Anesthesia was
induced with intramuscular xylazine (0.5−2.0 mg/kg) and ketamine
(10 mg/kg). After endotracheal intubation, V- and A-lines were
inserted, and anesthesia was maintained using isoflurane (1−1.5%,
100% O2 or 50/50 O2/N2O, l L/min). During anesthesia, heart rate,
respiration rate, blood pressure, O2 saturation, and end tidal CO2 were
monitored. The baboon was catheterized antecubitally for radiotracer
injection, and a radial arterial line was placed for metabolite analysis.
Brain imaging. PET-MR images were acquired in a Biograph mMR
scanner (Siemens, Munich, Germany) and PET compatible 8-channel
Baboon PET-MR Image Analysis. PET data were motion-corrected,
spatially smoothed with a 2.5 mm fwhm Gaussian filter, and registered
to the Black baboon brain atlas43 using JIP tools optimized for
nonhuman primate data processing (www.nitrc.org/projects/jip).
Image registration was carried out on high-resolution MPRAGE
MRI image using a 12 degree of freedom linear algorithm and a
nonlinear algorithm to the atlas brain. The transformation was then
applied to the simultaneously collected dynamic PET data.
Kinetic modeling was carried out in PMOD (PMOD3.3, PMOD
Technologies Ltd., Zurich, Switzerland). VOIs were selected according
to the Black baboon brain atlas. A common VOI mask was applied to
both baboon scans. TACs were exported from the whole brain,
cerebellum, primary motor cortex, putamen, thalamus, primary visual
cortex, caudate, and white matter VOIs for analysis. A two-tissue
compartmental model was used to estimate regional volume of
H
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distribution (VT) with a metabolite-corrected plasma TAC. Voxel-wise
VT maps were calculated using a Logan plot from the dynamic PET
data.44 The time until linearity of the plot was achieved and
determined for each scan.
In addition, regional K1/k2 and k3/k4 values were also obtained from
the baseline scans using a two-tissue compartmental model. It is
known that
Health under grant numbers R01DA030321 (J.M.H.; S.J.H)
with additional support under grant R01DA028301 (S.J.H.).
This research was carried out at the Athinoula A. Martinos
Center for Biomedical Imaging at the Massachusetts General
Hospital, using resources provided by the Center for Functional
Neuroimaging Technologies, P41EB015896, a P41 Regional
Resource supported by the National Institute of Biomedical
Imaging and Bioengineering (NIBIB), National Institutes of
Health. This work also involved the use of instrumentation
supported by the NIH Shared Instrumentation Grant Program
and/or High-End Instrumentation Grant Program; specifically,
grant nos: S10RR017208, S10RR026666, S10RR022976,
S10RR019933, and S10RR029495. The authors are grateful
to Joe Mandeville and Helen Deng as well as the Martinos
Center radiopharmacy and imaging staff (Grae Arabasz, Shirley
Hsu, Stephen Carlin, Chris Moseley, Nathan Schauer, Ehimen
Aisaborhale, Judit Sore) for help with nonhuman primate
experiments. HDAC activity screening was enabled by a Caliper
assay developed by Jennifer Gale and Yan-Ling Zhang at the
Broad Institute.
⎛
⎞
⎟
⎠
k3
k4
Bmax
Kd
K1
k2
=
⎜1 +
⎝
app
with the assumption that Kd
= 0.4 μM45 (assumed from ex vivo
HDAC-complex Kd measure for SAHA, which exhibits a similar IC50 in
in vitro activity-based assays we determined). Averaged Bmax from the
selected VOIs was estimated to be 19.6 2.9 μM (mean SD).
Plasma and Metabolite Analysis. Arterial samples collected during
imaging from the baboon were centrifuged to obtain plasma, which
was then removed and placed in an automated gamma counter that
was calibrated to the PET scanner. Metabolite analysis was conducted
on a custom automated robot fitted with Phenomenex SPE Strata-X
500 mg solid phase extraction cartridges that were primed with ethanol
(2 mL) and deionized water (20 mL). Protein precipitation was
achieved by addition of plasma (300 μL) to acetonitrile (300 μL),
which was centrifuged for 1 min to obtain protein-free plasma (PFP).
300 μL of PFP/acetonitrile solution was diluted into deionized water
(3 mL), loaded onto the C18 cartridge, and removed of polar
metabolites with 100% water. Next, a series of extractions were
performed using water and acetonitrile in quantities: 95:5, 90:10,
85:15, 80:20, 70:30, 60:40, 30:70, and 100% acetonitrile at a volume of
4 mL. A control experiment was performed before metabolite analysis
to determine the retention of the parent compound by injection of a
small amount of [11C]6 onto a test series of extraction cartridges. Each
sample was counted in a WIZARD2 Automatic Gamma Counter to
determine the presence of radiolabeled metabolites.
Brain Metabolite Analysis. One rat under pentobarbital anesthesia
(50 mg/kg, ip) was administered 0.538 mCi of [11C]6 by tail vein
injection in 0.7 mL and after 30 min, sacrificed by pentobarbital
overdose (additional 150 mg/kg, ip), and followed by rapid
decapitation. The brain was removed, sagitally hemisected, and
homogenized in 2 mL acetonitrile. The resulting suspension was
centrifuged at 1500 rpm for 5 min, and 0.6 mL of clear supernatant
was vortexed with 3 mL water and applied to Phenomenex SPE Strata-
X 500 mg solid phase extraction cartridges, washed, and fractionally
eluted with water containing increasing proportions of acetonitrile
(100:0, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, 30:70, 0:100). Each
sample was counted in a WIZARD2 Automatic Gamma Counter to
determine the presence of radiolabeled metabolites.
ABBREVIATIONS USED
■
HDACs, histone deacetylases; CNS, Center for Nanoscale
Systems; PET, positron emission tomography; PPB, plasma
protein binding; DAT, dopamine transporter; P-gp, P-
glycoprotein; NHP, nonhuman primate; GWAS, genome-
wide association studies; SNPs, single nucleotide poly-
morphisms; tPSA, total polar surface area; TAC, time−activity
curve
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ASSOCIATED CONTENT
* Supporting Information
■
S
The results of off-target binding, mPET imaging in rodents and
SAHA pretreated study in NHP were shown in Table S1,
Figure S1−S6. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: 617-726-6596; E-mail: hooker@nmr.mgh.harvard.
■
edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.W. and H.Y.W. are supported by the Harvard/MGH Nuclear
Medicine Training Program from the Department of Energy
(DE-SC0008430). Research was supported by the National
Institute of Drug Abuse (NIDA) of the National Institutes of
I
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