In vivo PET imaging of histone deacetylases by 18F- suberoylanilide hydroxamic acid (18F-SAHA)

Article abstract of DOI:10.1021/jm200620f

Histone deacetylases (HDACs) are a group of enzymes that modulate gene expression and cell state by deacetylation of both histone and non-histone proteins. A variety of HDAC inhibitors (HDACi) have already undergone clinical testing in cancer. Real-time i

Full text of DOI:10.1021/jm200620f

ARTICLE
pubs.acs.org/jmc
In Vivo PET Imaging of Histone Deacetylases by 18F-Suberoylanilide
Hydroxamic Acid (18F-SAHA)
J. Adam Hendricks,† Edmund J. Keliher,† Brett Marinelli, Thomas Reiner, Ralph Weissleder,* and
Ralph Mazitschek*
Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, 185 Cambridge Street,
Massachusetts 02114, United States
S Supporting Information
b
ABSTRACT: Histone deacetylases (HDACs) are a group of enzymes that modulate
gene expression and cell state by deacetylation of both histone and non-histone proteins.
A variety of HDAC inhibitors (HDACi) have already undergone clinical testing in cancer.
Real-time in vivo imaging of HDACs and their inhibition would be invaluable; however,
the development of appropriate imaging agents has remained a major challenge. Here, we
describe the development and evaluation of 18F-suberoylanilide hydroxamic acid (18F-
SAHA 1a), a close analogue of the most clinically relevant HDAC inhibitor suberoyla-
nilide hydroxamic acid (SAHA). We demonstrate that 1a has near identical biochemical
activity profiles to that of SAHA and report findings from pharmacokinetic studies. Using
a murine ovarian cancer model, we likewise show that HDAC inhibitor target binding
efficacy can be quantitated within 24 h of administration. 1a thus represents the first 18F-
positron emission tomography (PET) HDAC imaging agent, which also exhibits low
nanomolar potency and is pharmacologically analogous to a clinically relevant HDAC inhibitor.
’ INTRODUCTION
studies have now elucidated specific roles for different HDAC
isoforms in the regulation of fundamental biological processes.3
Notably, they have been demonstrated to be master regulators of
mitosis, cell differentiation, apoptosis,6 chromatin organization,7
metabolism,8 learning and memory,9 and the immune response.10
Given the relevance that most of these critical cellular functions
have to disease, small molecule modulators of chromatin mod-
ifying enzymes are now being developed, not only for oncological
applications but more broadly for the treatment of psychiatric,
immunological, neuromuscular, inflammatory, metabolic, genet-
ic, and infectious diseases.2,11,12
SAHA is a broad spectrum inhibitor that potently targets class
I and class II HDAC enzymes.13 At present, SAHA and other
HDACi are being explored as potential drug therapies for a
number of different malignancies, both as single agents and in
combination with other agents.2,12,14 A critical bottleneck in the
clinical development and use of HDACi has been the inability to
rapidly quantitate target inhibition in the clinic. Acetylation of
peripheral mononuclear cells initially showed early promise as a
potential biomarker but ultimately failed to correlate with clinical
response in patients.15 Conversely, conventional anatomic ima-
ging modalities used to monitor clinical response are only cap-
able of reporting on tumor size or on rudimentary physiologic
changes such as alterations in perfusion, both of which occur
relatively late after treatment onset. In vivo imaging of HDAC
Histone deacetylases (HDACs) are enzymatic components of
chromatin modifying complexes and modulate gene expression
via interactions with transcriptional regulatory proteins and via
modification of the amino-terminal histone tail lysine residues.1,2
More recently a considerable number of non-histone protein
substrates have also been identified, indicating that lysine acet-
ylation represents a post-translational modification with broad
regulatory significance.3 To date, 18 different HDACs have been
characterized and, based on their phylogeny, grouped into four
major classes.4 Class I (HDAC1ꢀ3,8), class II (HDAC4ꢀ7,9),
and class IV (HDAC11) are zinc-dependent hydrolases that are
formally referred to as HDACs. In contrast, class III (Sirt1ꢀ7)
enzymes, the sirtuins, function by a catalytically distinct, NAD+-
dependent mechanism. While the enzymatic activities of HDAC
isoforms overlap in vitro, their biological activity and substrate
selectivities in vivo are typically regulated by specific multiprotein
binding partners, as well as by their unique tissue and cellular
distribution.
The first HDAC inhibitors (HDACi) were developed for
cancer applications following findings that they induce apoptosis
and differentiation in malignant cell lines.5,6 Thus far, two
chemically distinct HDACi, suberoylanilide hydroxamic acid
(SAHA) 2 (Vorinostat, Merck Research Laboratories) and the
more recently developed microbial natural product FK228 3
(Romidepsin, Celgene),2 have received FDA approval for the
treatment of specific T-cell malignancies (Figure 1). In addition,
increasing information regarding HDAC function from genetic
Received: May 21, 2011
Published: July 01, 2011
r
2011 American Chemical Society
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Figure 1. Structures of HDAC inhibitors and HDAC imaging agents.
Scheme 1. Synthesis of 1ba
a (a) Methyl 8-chloro-8-oxooctanoate, Et3N, CH2Cl2, room temp, 1 h, 54%; (b) 50% NH2OH (aq), 1 N NaOH(MeOH), room temp, 12 h, 65%.
proteins (and to some degree their activity state) has likewise
been rudimentary at best and, as such, detailed analyses have
been limited to readily accessible tissue specimens, which can
only be analyzed using ex vivo techniques. Thus, in order to
investigate the efficiency of existing and novel HDACi, there is a
clear need for imaging probes. Moreover, imaging of HDACs
may provide important basic scientific insights, which could lead
to novel clinical applications.
specificity for individual HDAC isoforms. For example, the prin-
cipal enzymes that metabolize BLT are HDAC8 and (to a lesser
degree) class IIa HDACs. In contrast, current clinical HDAC
inhibitor candidates primarily target HDACs 1, 2, 3, and 6.13,19
’ RESULTS AND DISCUSSION
Here we describe the development of an in vivo PET imaging
agent with strong similarities in pharmacology, potency, and
isoform selectivity to SAHA, the most clinically relevant HDAC
inhibitor. Introduction of an 18F label to the 4-position of the
aromatic ring of SAHA (18F-SAHA 1a) appeared to be the most
practical approach. To first determine that this functionalization
would not negatively impact the compound’s biological activity,
we synthesized and profiled 19F-SAHA 1b against HDACs 1ꢀ9.
The synthesis was readily accomplished by following reported
strategies, with minor modification (Scheme 1).20 Synthesis
began with acylation of the commercially available 4-fluoroani-
line 7b, with suberic chloride methyl ester. Subsequent treatment
with basic hydroxylamine resulted in the conversion of the
methyl ester 8b to the hydroxamic acid.
The biochemical profiling of 1b against purified HDAC iso-
forms demonstrated that 1b, as expected, inhibited only HDACs
1ꢀ3 and 6.13 Moreover, inhibition of these primary HDAC
targets by 1b exhibited a potency and selectivity virtually indis-
tinguishable from SAHA itself. This finding thus identified 1b as a
promising candidate for the development of a PET imaging
probe (Figure 2a).
To date, only a few compounds for in vivo HDAC imaging
have been reported. Reid et al., for example, developed 6-
([18F]fluoroacetamide)-1-hexanoicanilide (FAHA) 4 for cere-
bral imaging. 4 is a 18F-functionalized hybrid of the HDAC
inhibitor SAHA, an acetyl lysine mimic that lacks the hydro-
xamate chelator, which is required for potent target binding.16
Furthermore, 4, which features a reactive fluoro ketone and
resembles an HDAC substrate rather than an inhibitor, is rapidly
metabolized in vivo, thus complicating imaging and analysis.
More recently the development of [11C]MS-275 5a, a carbon-11-
labeled version of the benzamide class HDAC inhibitor MS-275
(entinostat, Syndax Pharmaceuticals) 5b, has been reported for
cerebral imaging.17 5b, as most benzamide based HDAC inhibi-
tors, is selective for HDAC1ꢀ3, albeit 20- to 70-fold less potent
than SAHA.13 Unfortunately, the bloodꢀbrain barrier penetra-
tion of 5a was found to be unsuitable for the intended studies and
competition with cold 5b failed to demonstrate specific binding.17
Further, the short half-life of carbon-11 (∼20 min) compared to
fluorine-18 (∼110 min) imposes technical challenges and limits
potential applications. Another study by Ronen and co-workers
investigated the use of Boc-lysine trifluoroacetic acid 6 (BLT) as
a substrate for HDACs, using 19F magnetic resonance spectroscopy.18
Trifluoroacetyl-lysine-based substrates, however, are not suitable
for all HDACs because they exhibit large variations in their
Our next goal was to develop a reliable synthetic strategy for
the routine synthesis of 1a, which would enable generation of 1a
in quantities sufficient even for large animal studies. Direct
fluorination of 1,4-dinitrobenzene 9 with 18F (no carrier added
(nca)) under phase transfer conditions and microwave heating
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Figure 2. (a) Doseꢀresponse profile for 1b and 2 against HDACs 1ꢀ3 and 6. (b) Analytical radio-HPLC traces.
Scheme 2. Synthesis of 1aa
a (a) (1) K2CO3, K222,18Fꢀ (nca), DMSO, 120 °C, microwave, 5 min; (2) C18 isolation, MeOH elution, 78%; (b) (1) NaBH4, 10% Pd/C, room temp,
10 min; (2) LiChrolut EN isolation, THF elution, Na2SO4/Celite drying, 58%; (c) methyl 8-chloro-8-oxooctanoate, room temp, 5 min; (d) (1) 50%
NH2OH (aq), 1 N NaOH(MeOH), room temp, 3 min; (2) HPLC purification, concentration; (3) reconstitution into vehicle (88% two steps).
afforded 1-[18F]fluoro-4-nitrobenze 10 after 5 min at 120 °C.
This intermediate was consecutively reduced by catalytic hydro-
genation with sodium borohydride and Pd/C to yield 4-[18F]-
fluoroaniline 7a, which was isolated on a LiChrolut EN column.21
The 18F-labeled aniline was subsequently converted to the de-
sired product via compound 1a, following the same strategy
employed for synthesis of cold 1b using optimized conditions for
radiochemical synthesis (Figure 2b). Overall, this four-step syn-
thesis is remarkably clean, can be carried out in less than 2 h, and
affords 1a with a decay corrected yield of 40%. Following HPLC
purification, the radiochemical purity was greater than 98%
(Scheme 2). Several alternative routes attempting to introduce
the 18F later in the synthesis were explored unsuccessfully.
To date, very little information exists in the published scien-
tific literature on the pharmacokinetic and tissue distribution of
SAHA in murine models. Such knowledge, however, is critical
for the development of therapeutic applications in central nerv-
ous system disorders or spinal muscular atrophy. Thus, following
scale-up synthesis and characterization of the radiotracer, we next
tested the compound in vivo. Initially, we determined the plasma
half-life of 1a and its tissue distribution. The blood half-life, fol-
lowing a single bolus intravenous injection, was then measured
for three commonly utilized pharmacological formulations (A,
60% PEG, 40% water; B, 10% DMSO in water; C, 10% DMAC,
10% Solutol HS15, 80% PBS). All formulations exhibited a
biphasic pharmacokinetic profile, with rapid elimination during
the first 10 min. The initial t1/2 was estimated as less than 1ꢀ
4 min, mostly through renal clearance, and this was followed by a
terminal half-life that was approximately 10 times longer. No-
tably, the initial half-lives appeared to be strongly dependent on
the formulation, but these early time points are generally not
captured by traditional liquid chromatography/mass spectro-
metry (LC/MS) based approaches. Less than 1% of the dosed
compound wasstill present in whole blood when using formulation
A (60% PEG400, 40% water), while formulations B (10% DMSO,
90% water) and C (10% dimethylacetamide, 10% Solutol HS15,
80% PBS) resulted in retention of approximate 3% and 6% of the
compound, respectively (Figure 3a). Following this initial “burst”
phase, elimination half-lives appeared to be equivalent and cor-
related well with the blood-half-life, as determined by serial
in vivo PET imaging (Figure 3d). We also tested oral adminis-
tration of the compound (formulation C, Figure 3b) and ob-
served similar kinetics as determined by conventional HPLC
mass spectrometry.
We subsequently determined the tissue distribution of 1a. For
each experiment, cohorts of mice were sacrificed at 1 and 3 h
following intravenous injection of the probe. Tissues were then
excised and weighed, and 18F activity was measured. As shown
in Figure 3c, 1a had the highest concentration in the kidney,
liver and blood, with relatively low distribution in other tissues
(Figures 3c and 4a). Interestingly, cardiac tissue also appeared to
have comparatively low concentrations of 1a, which may in part
explain the relatively low cardiotoxicity of SAHA compared to
other HDACi. Of all organs investigated, the thymus and brain
exhibited the lowest concentration of 1a, implying that if used to
study brain function, SAHA would likely require intrathecal
administration.
Upon demonstrating that 1a exhibits a suitable pharmacolo-
gical profile, we next assessed whether administration of “cold”
SAHA could be used to measure HDAC inhibition in a mouse
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Figure 3. Blood half-life of 1a following (a) iv and (b) po administration; (c) biodistribution; (d) blood half-life determined by dynamic PET scan.
% ID/g is injected dose per gram of tissue. SUV, is standardized uptake value.
Figure 4. (a) Dynamic PETꢀCT following iv administration of 1a in C57BL/6 mouse. (b) PETꢀCT scan of tumor xenograft before and after
competition with “cold” SAHA, where blue dotted line indicates tumor perimeter. (c) Time dependent distribution of 1a.
model of cancer. Since SAHA is currently undergoing clinical
trials for the treatment of ovarian cancer, we selected the A2780
ovarian cancer model for this study. Consistent with class I
HDAC expression levels in both malignant and benign tissues
(Figure 4c), 1a was observed to accumulate in tumors in a time-
dependent manner, following its systemic administration. To
subsequently determine whether 1a imaging could be used to
measure HDAC inhibition in vivo, we performed repeat 1a
PET imaging experiments on a cohort of mice both before
and after cold SAHA treatment (2 ꢁ 2 mg injection, Figure 4b).
As expected for selective competition, the tumor/muscle ratio
decreased from 1.7 ( 0.12 to 1.3 ( 0.01 (23%), following two
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single doses of cold SAHA treatment in a 24 h period
(Figure 4b). These studies thus show proof of principle that 1a
imaging can be used to measure target engagement within 24 h of
drug administration. Overall, these studies describe the develop-
ment of a versatile HDAC PET imaging agent that not only
exhibits low nanomolar potency but is also pharmacologically
similar to a clinically relevant HDACi. It is likely that this
compound will be useful for testing SAHA and other HDACi
in the clinic.
Radiochemistry. [18F]Fluoride (nca) in 18O enriched water was
purchased from PETNET (Woburn, MA). Solid phase extraction
cartridges were purchased from Thermo (HYPERSEP C18, 500 mg,
3 mL) and Merck (LiChrolut EN, 200 mg, 3 mL). Preparative high per-
formance liquid chromatography (HPLC) purification (method A) was
achieved using a Machery-Nagel Nucleodur C18 Pyramid 250 mm ꢁ
10 mm Vario-Prep column (80:20 (v/v) in water/acetonitrile (MeCN)
with 0.1% trifluoroacetic acid at 5.5 mL minꢀ1), and analytical HPLC
3
(method B) was performed employing a Grace VYDAC (218TP510)
C18 reversed-phase column (eluents A = 0.1% TFA (v/v) in water and
B = MeCN; gradient 0ꢀ17 min, 5ꢀ60% B; 17ꢀ21 min, 60ꢀ95% B;
21ꢀ24 min, 95% B; 24ꢀ25 min, 95ꢀ5% B; 25ꢀ30 min, 5% B; 2 mL
minꢀ1) with a dual-wavelength UVꢀvis detector followed by a flow-
through γ detector connected in series. Preparative and analytical HPLC
analyses of 18F-labeled compounds were calibrated with the correspond-
ing 19F analogues. Microwave synthesis was conducted in a CEM
(Matthews, NC) microwave synthesizer operated with Discover soft-
ware package. Radioactive samples injected into mice were measured
using a Capintec 127R dose calibrator. Radioactivity in blood half-life
and biodistribution studies was quantified using a Perkin-Elmer Wallac
Wizard 3 in. 1480 automatic γ counter.
’ EXPERIMENTAL METHODS
Chemistry. Reagents were purchased from Acros, Alfa-Aesar, and
Sigma-Aldrich Co. and used without further purification unless other-
wise noted. Silica gel 60 was used for purification, 40ꢀ63 μm. Proton
and carbon nuclear magnetic resonance (1H and 13C NMR) spectra
were recorded on a Varian AS-400 (400 MHz) spectrometer. Chemical
shifts for protons are reported in parts per million (ppm) and are
referenced against the dimethylsulfoxide lock signal (1H, 2.50 ppm; 13C,
39.52 ppm). Data are reported as follows: chemical shift, integration,
multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), and
coupling constants (Hz). Preparatory and analytical LC/MS were
performed on mass directed autopurification systems from Waters Co.
(Milford, MA), operated by Fractionlynx 4.0 or Masslynx software with
Waters Xterra columns (C18, 10 μm, 19 mm ꢁ 50 mm for preparative;
C18, 5 μm, 4.6 mm ꢁ 50 mm for analytical). High-resolution mass
spectra were acquired on a Bruker Daltonics APEXIV 4.7 T Fourier
transform ion cyclotron resonance mass spectrometer (FT-ICR-MS),
with ESI (electrospray ion) source. The chemical purity of the target
compounds was determined by LC/MS using the following conditions:
a Waters SFO/3100 with Waters Xterra C18 column using a binary
solvent system (0.1% TFA in water and acetonitrile), with monitoring
between 200 and 400 nm. The purity of each compound was g95%.
7-(4-Fluorobenzoylamino)heptanoic Acid Methyl Ester
8b. 4-Fluoroaniline 7b (259 μL, 2.7 mmol) and triethylamine
(376 μL, 2.7 mmol) were dissolved in DCM (2 mL) and cooled to 0 °C.
To this solution was added methyl suberyl chloride (350 μL, 2.5 mmol).
After 1 h the mixture was diluted with DCM (10 mL) and then washed
with H2O (10 mL), brine (10 mL) and then dried with magnesium sul-
fate. Purification via column chromatography (silica gel, DCM/EtOAc
40%) gave product 8b (383 mg, 54%). 1H NMR (400 MHz, DMSO)-
d6) δ 9.91 (s, 1H), 7.59 (dd, J = 9.2, 5.1 Hz, 2H), 7.12 (t, J = 9.0 Hz, 2H),
3.57 (s, 3H), 2.28 (q, J = 7.5 Hz, 4H), 1.63ꢀ1.44 (m, 4H), 1.29 (dd, J =
7.3, 3.6 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 173.37, 171.09,
157.778 (d, JCꢀF = 240.4 Hz), 135.77, 120.69 (d, JCꢀF = 8.1 Hz),
115.20 (d, JCꢀF = 22.2 Hz), 51.21, 36.24, 33.24, 28.25, 24.94, 24.34.
HRMS (ESI) for C15H20FNO3: calculated 282.1500; observed 282.1506
[M + H]+, 304.1332 [M + Na]+.
Synthesis of 1a. A 10 mL microwave test tube was charged with
26.9 ( 2.7 mCi (1.0 GBq) [18F]fluoride (nca), K2CO3 (1.38 mg,
10 μmol) in water, 135 μL, Kryptofix 222 (6.8 mg, 18 μmol), and 1 mL
of MeCN. Water was removed azeotropically by microwave heating to
98 °C and a flow of argon. Azeotropic drying is repeated by the addition
of anhydrous MeCN (4 ꢁ 1 mL) at 3 min intervals. After the mixture
wascooledtoroom temperature, 1,4-dinitrobenzene9 (4 mg, 23.8 μmol)
in DMSO (500 μL) was added. The vessel was sealed and heated to
120 °C for 5 min and then cooled to room temperature. The reaction
mixture was diluted with water (8 mL) and passed through a C18 cart-
ridge (Thermo, 500 mg, conditioned with 10 mL of EtOH and 2 ꢁ
10 mL of water) trapping the 4-[18F]fluoronitrobenzene (10). The C18
cartridge was washed with water (5 mL), and 16.1 ( 0.9 mCi (596 MBq)
10 was eluted from the cartridge with MeOH (1 mL) in 78.3 ( 6.4%
dcRCY. A 10 μL aliquot was removed for HPLC analysis (method B), 10
tR = 21.4 min. After addition of 3 mg of Pd/C and 28 mg (740 μmol) of
NaBH4, the reaction mixture was stirred at room temperature for 5 min,
at which time unreacted NaBH4 was quenched by the addition of 300 μL
of 6 N HCl. The mixture was diluted with 1 N NaOH(aq) (8 mL) and
passed through a Lichrolut EN cartridge (Merck, 500 mg, conditioned
with 10 mL of EtOH and 2 ꢁ 10 mL of water). 4-[18F]Fluoroanaline 7a
was eluted from the LiChrolut EN cartridge and through 500 mg of
Na2SO4 and Celite (3:2 w/w) with THF (1 mL) to give 10.1 ( 1.3 mCi
(370 MBq) in 57.5 ( 5.0% dcRCY. A 10 μL aliquot was removed for
HPLC analysis (method B), 7a tR = 11.4 min. To the 7a/THF solution
was added methyl 8-chloro-8-oxooctanoate (34 μL, 242 μmol), and the
mixture was stirred for 5 min at room temperature. A 10 μL aliquot was
removed for HPLC analysis (method B), 8a tR = 22.8 min. The addition
of 250 μL of 50% NH2OH(aq) and 750 μL of 1 N NaOH(MeOH) and
stirring for 3 min provided the desired hydroxamic acid 1a. A 10 μL
aliquot was removed for HPLC analysis (method B), 1a tR = 17.0 min.
THF and MeOH were evaporated off (50 °C microwave heating under a
stream of argon), and the reaction mixture was purified by preparative
HPLC (method A), providing 5.1 ( 0.7 mCi (189 MBq) 1a in 120 (
12 min, a 39.5 ( 6.0% dcRCY in 97.0 ( 4.7% radiochemical purity over
4 steps and HPLC purification.
N-Hydroxy-N0-(4-fluorophenyl)octanediamide 1b. Methyl
ester 8b (180 mg, 0.640 mmol) was suspended in a 1:1 (v/v) mixture of
methanol and 50% hydroxylamine (aq). To this suspension was added
1 N NaOH(aq) (1.5 mL). After 12 h the mixture had become homo-
geneous. Then 1 N HCl was added, bringing the solution back to a
neutral pH, upon which the product precipitated from solution. Filtra-
tion of the white solid afforded the product 6 (84 mg, 65%). 1H NMR
(400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.92 (s, 1H), 8.67 (s, 1H), 7.59
(dd, J = 9.2, 5.1 Hz, 2H), 7.12 (t, J = 8.9 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H),
1.93 (t, J = 7.4 Hz, 2H), 1.55 (t, J = 14.5, 7.2 Hz, 2H), 1.47 (t, J = 14.2, 7.2
Hz, 2H), 1.31ꢀ1.20 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ
Mice. Five Nu/Nu mice received subcutaneous injections with 106
A2780 human ovarian cancer cells in Matrigel into each flank and were
imaged 10 days later. Mice were anesthetized (Isoflurane 1.5%, O2 2 L/
min) during PETꢀCT imaging. Therapeutic doses of SAHA (2 mg in
100 μL of 60% PEG400 40% water) were administered ip following
initial PETꢀCT scan and repeated 24 h later. After 48 h from initial
therapeutic dose, PETꢀCT scans were performed again following the iv
171.11, 169.09, 157.78 (d, JCꢀF = 240.4 Hz), 135.77, 120.70 (d, JCꢀF
=
8.1 Hz), 115.21 (d, JCꢀF = 22.2 Hz), 36.29, 32.26, 28.43, 25.06, 25.03.
HRMS (ESI) for C14H19FN2O3: calculated 283.1452; observed
283.1444 [M + H]+, 305.11258 [M + Na]+.
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administration of 1a. Ten C57BL/6 mice were used for blood half-life
studies. Mice were administered 243 ( 25 μCi of 1a by tail vein iv
injection formulated in 60% PEG400 in water (n = 3), 10% DMSO in
water (n = 3), 20% DMAC.Solutol (1:1) in saline (n = 3). In a separate
experiment a mouse (n = 1) was administered 265 μCi 1a by po. Blood
sampling was performed by retro-orbital bleed using tared capillary
tubes. Samples were weighed, and activity was measured by γ counter.
Six C57BL/6 mice were injected with 241 ( 18 μCi 1a by tail vein iv
injection and used to determine biodistribution. Animals were sacrificed
at 1 h (n = 3) and 3 h (n = 3) after injection. Tissues were excised and
weighed, and activity was measured. Graphpad Prism 4.0c (GraphPad
Software Inc., San Diego, CA) was used for regression and statistical
analysis. All animal experiments were approved by the Massachusetts
General Hospital Institutional Review Committee.
’ ABBREVIATIONS USED
BLT, Boc-lysine trifluoroacetic acid; DCM, dichloromethane;
DMSO, dimethylsulfoxide; DMAC, dimethylacetamide; FAHA,
6-([18F]fluoroacetamide)-1-hexanoicanilide; HDAC, histone
deacetylase; HDACi, histone deacetylase inhibitors; HPLC,
high-performance liquid chromatography; LC/MS, liquid chro-
matographyꢀmass spectrometry; nca, no carrier added; PEG, poly-
ethylene glycol; PBS, phosphate buffered saline; PET, positron
emission tomography; PETꢀCT, positron emission tomographyꢀ
computed tomography; SAHA, suberoylanilide hydroxamic acid;
SUV, standardized uptake value; THF, tetrahydrofuran
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reconstructed from 600 million coincidental 511 keV photon counts
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Counts were rebinned in 3D by registering photons spanning no more
than three consecutive rings, then reconstructed into sinograms by
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back-projection algorithm, with a Ramp filter at a Nyquist cutoff of 0.5.
Image pixel size was anisotropic, with dimensions of 0.796 mm in the z
direction and 0.861 mm in the x and y directions for a total of 128 ꢁ
128 ꢁ 159 pixels. Calibration of PET signal preceded all scans and was
done by scanning an 8.0 cm cylindrical phantom containing a known
amount of 18F isotope.
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CT was reconstructed from 360 projections of X-rays with a cone
beam angle of 9.3° over 360° perpendicular to the animal bed. 80 keV
X-rays were transmitted from a 500 μA anode source 347 mm from the
center of rotation and recorded on a CCD detector, containing 2048
transaxial and 3072 axial pixels. Projections were calibrated using 70 dark
and 70 light images, interpolated bilinearly, processed through a Sheppꢀ
Logan filter, then reconstructed using a filtered back projection algo-
rithm. Isotropic CT pixel size was 110.6 μm, with a total of 512 ꢁ 512 ꢁ
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’ ASSOCIATED CONTENT
S
Supporting Information. NMR data for compounds 1b
b
and 8b. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For R.W.: phone, 617-643-6133; e-mail, rweissleder@mgh.
harvard.edu. For R.M.: phone, 617-643-6286; e-mail, rmazitschek@
mgh.harvard.edu.
Author Contributions
†These authors contributed equally.
(10) de Zoeten, E. F.; Wang, L.; Butler, K.; Beier, U. H.; Akimova, T.;
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Hancock, W. W. Histone deacetylase 6 and heat shock protein 90
control the functions of Foxp3(+) T-regulatory cells. Mol. Cell. Biol.
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Hancock, W. W. Immunomodulatory effects of deacetylase inhibitors:
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’ ACKNOWLEDGMENT
Financial support from the NCI (Grants P50CA086355,
T32CA079443, and U24CA092782) is gratefully acknowledged.
T.R. received a stipend from the Deutschen Akademie der
Wissenschaften Leopoldina (Grant LPDS 2009-24).
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