Radiosynthesis and preliminary evaluation of an 18F-labeled tubastatin A analog for PET imaging of histone deacetylase 6

Article abstract of DOI:10.1002/jlcr.3823

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family because of its characteristics, namely, its cytoplasmic localization and ubiquitin binding. HDAC6 has been implicated in cancer metastasis and neurodegeneration. In the present study, we performed radiosynthesis and biological evaluation of a fluorine-18–labeled ligand [¹⁸F]3, which is an analog of the HDAC6-selective inhibitor tubastatin A, for positron emission tomography (PET) imaging. [¹⁸F]3 was synthesized by a two-step reaction composed of ¹⁸F-fluorination and formation of a hydroxamic acid group. IC₅₀ values of 3 against HDAC1 and HDAC6 activities were 996 nM and 33.1 nM, respectively. A biodistribution study in mice demonstrated low brain uptake of [¹⁸F]3. Furthermore, bone radioactivity was stable at around 2% ID/g after injection, suggesting high tolerance to defluorination. Regarding metabolic stability, 70% of the compound was observed as the unchanged form at 30 minutes post injection in mouse plasma. A small animal PET study in mice showed that pretreatment with cyclosporine A had no effect on initial brain uptake of [¹⁸F]3, suggesting low brain uptake of [¹⁸F]3 was not caused by the P-glycoprotein–mediated efflux. While PET imaging using [¹⁸F]3 has a limitation with respect to neurodegenerative diseases, further studies evaluating its utility for certain cancers are worth evaluating.

Full text of DOI:10.1002/jlcr.3823

Tago Tetsuro (Orcid ID: 0000-0002-9545-0922)
18
Radiosynthesis and preliminary evaluation of an F-labeled Tubastatin A analogue for
PET imaging of histone deacetylase 6
Tetsuro Tago, Jun Toyohara, Kenji Ishii
Research Team for Neuroimaging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-
cho, Itabashi-ku, Tokyo 173-0015, Japan.
Corresponding author: Jun Toyohara, Ph.D.
Research Team for Neuroimaging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-
cho, Itabashi-ku, Tokyo 173-0015, Japan
Tel: +81-3-3964-3241; Fax: +81-3-3964-1148
toyohara@pet.tmig.or.jp
Short title: Fluorine-18-labeled HDAC6 ligand
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Abstract (199/200 words)
Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family because of its
characteristics, namely its cytoplasmic localization and ubiquitin binding. HDAC6 has been
implicated in cancer metastasis and neurodegeneration. In the present study, we performed
radiosynthesis and biological evaluation of a fluorine-18-labeled ligand [18F]3, which is an
analogue of the HDAC6-selective inhibitor Tubastatin A, for PET imaging. [18F]3 was
synthesized by a two-step reaction composed of 18F-fluorination and formation of a
hydroxamic acid group. IC50 values of 3 against HDAC1 and HDAC6 activities were 996 and
33.1 nM, respectively. A biodistribution study in mice demonstrated low brain uptake of [18F]3.
Furthermore, bone radioactivity was stable at around 2% ID/g after injection, suggesting high
tolerance to defluorination. Regarding metabolic stability, 70% of the compound was observed
as the unchanged form at 30 min post injection in mouse plasma. A small animal PET study in
mice showed that pre-treatment with cyclosporine A had no effect on initial brain uptake of
[18F]3, suggesting low brain uptake of [18F]3 was not caused by the P-glycoprotein-mediated
efflux. While PET imaging using [18F]3 has a limitation with respect to neurodegenerative
diseases, further studies evaluating its utility for certain cancers are worth evaluating.
Keywords: Histone deacetylase 6, Positron emission tomography, Tubastatin A, Fluorine-18
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1. INTRODUCTION
Histone deacetylases (HDACs) are enzymes that catalyze the deacetylation of acetylated lysine
residues of histones, which are the major components of chromatin.1 The HDAC family has
been classified into five classes according to their homology to yeast HDACs: class I (HDAC1,
2, 3, and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and 10), class III (sirtuins
(SIRT1–7)), and class IV (HDAC11).2-4 Except for sirtuins, which have nicotinamide adenine
dinucleotide-dependent catalytic activity, HDACs have a zinc dependent catalytic site.5
Acetylation of lysine residues in the N-termini of histones neutralizes their positive charge,
which weakens the interaction between histones and negatively charged DNA, thereby
allowing enhanced accessibility of transcription factors to the DNA.6 Abnormalities in such
epigenetic regulatory system are important factors in the development of diseases including
cancer and neurodegeneration; therefore, HDACs are considered promising therapeutic targets
for these diseases.7-11 For instance, suberoylanilide hydroxamic acid (SAHA, also known as
vorinostat) is a non-selective HDAC inhibitor that is the first US Food and Drug
Administration-approved hydroxamic acid-based drug for use in patients with cutaneous T-cell
lymphoma.3
HDAC6, which belongs to the class IIb, is a unique member because of its characteristic
localization and functions. HDAC6 has two nuclear exclusion domains and one cytoplasmic
anchoring domain, rendering it primarily localized in the cytoplasm.12 HDAC6 also has two
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catalytic domains with different substrate specificities,13 and catalyzes the deacetylation of
non-histone proteins such as α-tubulin, HSP90, cortactin, and the microtubule-associated
protein tau.14,15 Deacetylation of α-tubulin in microtubules increases cell motility and enhances
cancer cell metastasis and invasion.16,17 Additionally, HDAC6 has an ubiquitin binding domain
and is involved in the degradation of misfolded and ubiquitinated proteins.18,19 HDAC6 also
has a binding domain against the cytoskeletal motor protein dynein and couples ubiquitinated
proteins to dynein motors, thereby mediating their transport along microtubules to
aggresomes.20,21 These characteristics of HDAC6 have spurred interest in targeting it with
therapeutics for cancers and neurodegenerative diseases,14,22 with several clinical trials of
selective HDAC6 inhibitors for cancers having been performed.23
In the past decade, a number of radiolabeled HDAC ligands for positron emission tomography
(PET) imaging have been developed.24-26 Besides the detection of regions with abnormalities
in HDAC expression levels, PET imaging can contribute to the development of HDAC
inhibitor drugs by enabling the assessment of target engagement.27,28 The majority of HDAC
radioligands, including the first SAHA-based HDAC radioligand [18F]FAHA, are hydroxamic
acid-based with others being benzamide-based or carboxylic acid-based.24,29 In most cases,
HDAC radioligands show poor blood-brain barrier (BBB) permeability in rodents and non-
human primates, probably due to the high-polarity of the hydroxamic acid group.24 However,
Hooker et al. reported that hydroxamic acid-based radioligands containing an adamantane
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group, which is often conjugated with drugs to improve their brain penetrance,30,31 readily
crossed the BBB in rodents and non-human primates.32,33 In particular, the carbon-11 labeled
ligand [11C]Martinostat which has high binding affinity for class I HDACs underwent a first-
in-human study to evaluate neuroepigenetic regulation.34 As expected, [11C]Martinostat
demonstrated excellent uptake in the human brain, and higher uptake was observed in the
cortical gray matter compared with white matter. Furthermore, [11C]Martinostat PET was also
used to image HDAC dysregulation in patients with schizophrenia.35
Due to the characteristics of HDAC6, selective imaging of HDAC6 could facilitate research
into its roles in cancers and neurodegenerative diseases. Regarding the development of HDAC6
selective radioligands, Lu et al. reported on 11C-carbonylation methods for labeling Tubastatin
A, an HDAC6 selective inhibitor (1 in Figure 1),36 that employed [11C]carbon monoxide.37
Radiosynthesis of the Tubastatin A analogue [11C]KB631 ([11C]2 in Figure 1) with [11C]methyl
iodide was also reported by Lu et al.,38 and its preclinical evaluation for visualization of
B16F10 melanoma has recently been reported by Vermeulen et al.39 Additionally,
[18F]Bavarostat was developed by Strebl et al.40 as an adamantane-conjugated HDAC6
selective radioligand. Although [18F]Bavarostat needs a unique ruthenium-mediated
18F‑deoxyfluorination41 for its radiosynthesis, its BBB permeability and specific binding in the
brain was confirmed by in vivo blocking studies in non-human primates.
Here, we report the development of a novel 18F-labeled Tubastatin A analogue [18F]3 (Figure
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1) as an HDAC6 selective PET imaging ligand. We expected that the basic structure of
Tubastatin A analogues was acceptable for structural modification for labeling with fluorine-
18, which has a relatively long half-life (109 min) that allows centralized production of
radiopharmaceuticals, because the relationship between the structures of the analogues and
their HDAC6 selectivity were well known.36,42 The chemical structure of 1 consists of three
motifs: a hydroxamic acid zinc-binding group that localizes a zinc ion within the HDAC active
site, a carboline-based cap group that interacts with the protein surface of the binding pocket,
and a benzyl linker group that bridges the two groups. The benzyl linker and carboline cap
groups contribute to the selectivity for HDAC6, which has a wider and shallower catalytic
channel compared with that of HDAC1. Kalin et al. demonstrated that a substituent could be
introduced at the nitrogen atom of tetrahydropyridine of the β-carboline regioisomer of 1
without seriously disrupting HDAC6 selectivity.42 Therefore, we decided to substitute the
methyl group of 2 with a fluoroethyl group for 18F-labeling of the Tubastatin A analogue.
Radiosynthesis of [18F]3 was achieved using a two-step reaction composed of 18F-fluorination
by general nucleophilic substitution and conversion of a methylester group into a hydroxamic
acid group. We intended to perform this radiosynthesis without intermediary solid-phase
extraction or high-performance liquid chromatography (HPLC) purification steps, which have
been incorporated in other hydroxamic acid-based radioligands,32,40,43 because these processes
often complicate automated radiosynthesis. In addition, preclinical evaluations such as binding
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selectivity assays and biodistribution assays were performed to assess the potential utility of
[18F]3 for visualization of HDAC6.
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2. EXPERIMENTAL
2.1. General
An aqueous solution of tetrabutylammonium hydrogen carbonate (0.075 M) was purchased
from ABX GmbH (Radeberg, Germany). Other reagents were purchased from Sigma-Aldrich
(St. Louis, MO), Tokyo Chemical Industry (Tokyo, Japan), or FUJIFILM Wako Pure Chemical
(Osaka, Japan). Automated flash chromatography was performed using an EPCLC AI-580
1
(Yamazen Science, Osaka, Japan). H-Nuclear magnetic resonance (NMR) spectra were
recorded on a JEOL Model ECP-400 (400 MHz; JEOL, Tokyo, Japan) or a Bruker AVANCE
III HD 400 (400 MHz; Bruker, Billerica, MA). Chemical shifts (δ) are relative to TMS (0 ppm)
and coupling constants are expressed in Hertz (Hz). Multiplicity was defined as following
abbreviations: s = singlet, d = doublet, t = triplet, dd = double doublet, m = multiplet, br =
broad. Electrospray ionization-mass spectrometry (ESI-MS) was measured using an LCT
Premier XE (Waters, Milford, MA). High-resolution mass spectrometry (HRMS) (ESI) was
carried out with a Q Exactive equipped with an UltiMate 3000 HPLC system (Thermo Fisher
Scientific, Waltham, MA).
2.2. Chemistry
Methods for the synthesis and characterization of non-radioactive 2, compounds shown in
Scheme 1, and the precursor of [11C]2 are described in detail in the Supporting Information.
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2.3. Radiosynthesis
A method for the radiosynthesis of [11C]2 is described in the Supporting Information.
Automated radiosynthesis, purification, and formulation of the [18F]3 injection solution were
performed using a modified F100 synthesizer (Sumitomo Heavy Industries, Tokyo, Japan).
[18F]Fluoride was produced by proton irradiation of 18O-enriched water (Taiyo Nippon Sanso,
Tokyo, Japan) at 50 μA for 15 min using an HM-20 cyclotron (Sumitomo Heavy Industries).
The irradiated 18O-enriched water containing [18F]fluoride was passed through a Sep-Pak
Accell Plus QMA Plus Light cartridge (Waters), and the trapped [18F]fluoride was eluted with
a mixture of 0.075 M tetrabutylammonium hydrogen carbonate (0.5 mL) and acetonitrile (0.5
mL) to a reaction vessel. After the solvent was dried azeotropically, 1 mL of dry acetonitrile
was added, and azeotropic drying was repeated. A mixture of precursor (8) in acetonitrile (2
mg/700 μL) was added to the reaction vessel, and the reaction mixture was heated at 90 °C for
15 min. After removal of the reaction solvent by heating at 90 °C under a slow helium stream,
the reaction vessel was cooled to 30 °C. A mixture of 50% hydroxylamine aqueous solution
(0.1 mL) and 2 M sodium hydroxide in methanol (0.2 mL) was added to the residue and stirred
at the same temperature for 10 min. Thereafter, 0.5 M hydrogen chloride (1.2 mL) was added
and the reaction mixture was purified by semi-preparative HPLC equipped with a radioactivity
detector system [Column: YMC-Pack Pro C18 RS, 5 μm, 10×250 mm (YMC, Kyoto, Japan);
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Eluent: acetonitrile/25 mM ammonium formate (adjusted to pH 4.7 with formic acid) = 30/70;
Flow rate: 4.5 mL/min; UV: 260 nm]. The fraction containing the product was combined with
250 mg/mL ascorbic acid injection solution (0.2 mL) and the solvent was removed by
evaporation. Finally, the residue was formulated in physiological saline and the product was
analyzed with an analytical HPLC [Column: Sunniest C18, 5 μm, 4.6×150 mm (ChromaNik
Technologies, Osaka, Japan); Eluent: acetonitrile/water/trifluoroacetic acid in water =
27.5/72.5/0.1; Flow rate: 1.0 mL/min; UV: 254 nm]. Representative semi-preparative HPLC
and analytical HPLC chromatograms are shown in the Supporting Information.
2.4. Log D determination
A saline solution containing [18F]3 (300 kBq) was added to a centrifuge tube containing 3 mL
of 1-octanol and 3 mL of Dulbecco's phosphate-buffered saline. The centrifuge tube was
vortexed for 20 sec and rested for 10 sec; this procedure was repeated twice. The 1-octanol and
aqueous phases were separated by centrifugation (720×g for 3 min at rt). An aliquot of the 1-
octanol phase (2 mL) was transferred into a centrifuge tube containing 1 mL of 1-octanol and
3 mL of Dulbecco's phosphate-buffered saline, and the previous mixing and centrifugation
procedures were repeated. These processes, from the transfer of the 1-octanol phase to
centrifugation, were repeated once more. Aliquots (100 μL) were collected from both the 1-
octanol and aqueous phases, and the radioactivity of each sample was measured with a gamma
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counter (2480 Wizard2; PerkinElmer, Waltham, MA). The log D value was determined using
the following equation: log D = log(radioactivity of 1-octanol sample/radioactivity of aqueous
sample).
2.5. In vitro HDAC enzyme inhibition assay
Binding affinities of 2 and 3 for HDAC1 and HDAC6 were measured using HDAC1 and
HDAC6 Fluorogenic Assay Kits (BPS Bioscience, San Diego, CA), respectively, following the
protocol described in the product datasheet. IC50 values were determined from dose-response
curves using GraphPad Prism software (GraphPad Software, San Diego, CA).
2.6. Ex vivo biodistribution assay in normal mice
All experiments using mice were approved by the Animal Experiment Committee of Tokyo
Metropolitan Institute of Gerontology and carried out in accordance with its animal
experimental protocols (Authorization number: 17081 and 18013). Asaline solution containing
[11C]2 (4.8 MBq/200 μL) or [18F]3 (0.77 MBq/200 μL) was administered to ddY mice (8 week-
old, male, 33.1 ± 1.8 g; Japan SLC, Hamamatsu, Japan; n = 4 per time point) via the tail vein.
The mice were killed by decapitation at 1, 5, 10, 30, and 60 min post-injection, and the organs
of interest were collected. After the samples were weighed, the radioactivity was counted with
a gamma counter (1282 CompuGamma CS, LKB Wallac, Turku, Finland).
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2.7. Metabolite analysis of [18F]3 in mice
A saline solution of [18F]3 (37 MBq) was injected to ddY mice (8 week-old, male; Japan SLC;
n = 3 per time point) via the tail vein. The mice were killed by decapitation at 15 and 30 min
post-injection, and cardiac blood was collected using a heparinized syringe and kept on ice.
Plasma fractions were separated from the blood by centrifugation (7,000×g for 1 min at 4 °C).
The plasma (200 μL) was deproteinized by addition of a 2-equivalent volume (400 μL) of
acetonitrile followed by centrifugation under the same condition. After the supernatant was
collected, the precipitate was resuspended with 400 μL of acetonitrile followed by
centrifugation under the same condition. This procedure was repeated twice, ultimately
resulting in an extraction efficacy of 94%. The combined supernatants were centrifuged
7,000×g for 3 min at 4 °C. After the precipitate was removed, the supernatant was combined
with 1 mLof 25 mM ammonium formate aqueous solution and analyzed using HPLC (Column:
YMC-Pack ODS-A, 5 μm, 10×250 mm; Eluent: acetonitrile/25 mM ammonium formate =
36/64; Flow rate: 3.0 mL/min; UV: 254 nm). Fractions were collected at 24-s intervals, and
their radioactivity was measured using a gamma counter (1282 CompuGamma CS).
2.8. Analysis of effect of P-glycoprotein (P-gp) inhibition of [18F]3 brain uptake in mice
A PET study in mice was performed using a small animal PET scanner (MIP-100, Sumitomo
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Heavy Industries).44 Eight week-old ddY mice (male, 35.4 ± 0.9 g; Japan SLC; n = 4 for each
group) received intravenous injection of 25 mg/kg of cyclosporine A (CsA) (4 mg/mL in
physiologic saline; Novartis, Basel, Switzerland). Control mice were treated with 6.3 mL/kg of
physiologic saline. Thirty minutes after the pre-treatment, the mice were given 17.7 ± 1.6 MBq
of [18F]3 via the tail vein under anesthesia with 1.5 % (v/v) isoflurane, and a 60-min dynamic
scan was performed immediately after the injection. The resulting sinograms were
reconstructed into 21 frames (8 × 30 s, 3 × 60 s, 2 × 120 s, 2 × 180 s, 3 × 300 s, 2 × 540 s, 1 ×
600 s) using an interactive reconstruction algorithm (3-dimensional ordered-subset expectation
maximization, provided by Sumitomo Heavy Industries; 1 iteration, 32 subsets). The final data
sets consisted of 31 slices, with a slice thickness of 0.85 mm and an in-plane image matrix of
256 × 256 pixels (0.3 × 0.3 mm pixel size). The data sets were fully corrected for random
coincidences and scatter. Standardized uptake value images were obtained by normalizing
tissue radioactivity concentrations according to the injected dose and body weight using PMOD
software (PMOD Technologies, Zurich, Switzerland). Regions of interest were drawn over the
whole brain to calculate brain uptake and generate time-activity curves.
2.9. Statistical analysis
The biodistribution and organ/blood ratios of radioligands in normal mice were statistically
analyzed by multiple t-tests corrected by the Holm-Šídák method using GraphPad Prism
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Software.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The synthetic route for reference compound 3 and chlorinated 18F-labeling precursor 8 are
shown in Scheme 1. To introduce a fluoroethyl moiety, 2-fluoroethyl p-toluenesulfonate (4)
was prepared by monofluorination of ethylene di(p-toluenesulfonate) with
tetrabutylammonium fluoride (TBAF). Compound 5 was synthesized according to a published
method from 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole.42 For synthesis of 3, 5 was N-
fluoroethylated with 4, and then a methylester group was converted to a hydroxamic acid group
with hydroxylamine. For synthesis of 8, 5 was N-hydroxyethylated with 2-bromoethanol and
then chlorinated with thionyl chloride. Initially, we attempted to synthesize a tosylated
precursor from 5 with ethylene di(p-toluenesulfonate) under the same reaction condition used
for the synthesis of 6, but formation of a dimerized by-product, which was bridged with an
ethylene group, was mainly observed. We also tried tosylation of 7 using tosyl chloride with
triethylamine or N,N,N’,N’-tetramethyl-1,3-propanediamine as a base,45 but we failed to obtain
the desired product. A fluorination test of 8 using TBAF in acetonitrile under heating
demonstrated formation of 6 (data not shown), therefore we proceeded to radiosynthesis of
[18F]3 using 8 as a precursor.
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3.2. Radiosynthesis of [18F]3
Radiosynthesis of [18F]3 was achieved with a two-step reaction (Scheme 2). The radiochemical
yield after HPLC purification of [18F]3 was 13 ± 4% (n = 3). The radiochemical purity and
molar activity of the [18F]3 saline solution at the end of synthesis were 97.2 ± 0.3% and 185 ±
56 GBq/μmol, respectively. The total synthesis time was 80–90 min from the end of
bombardment.
3.3. Lipophilicity determination
We determined the lipophilicity of [18F]3 using the shake-flask method, and the log D value
was 2.0, which is within the range of preferable lipophilicity for crossing the BBB.46
3.4. HDAC6 selectivity
Binding selectivity of compounds 2 and 3 was evaluated by inhibition assays against HDAC1,
as a representative HDAC, and HDAC6 (Figure 2 and Table 1). As shown in Figure 2,
inhibition curves for HDAC1 and HDAC6 of the fluoroethyl analogue 3 were almost identical
to those of 2, with an IC50 value of 33.1 nM toward HDAC6 and a selectivity of approximately
30-fold over HDAC1. These results were consistent with our expectation, which was that
introducing a fluoroethyl group to the nitrogen atom of the β-carboline structure would have a
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negligible effect on HDAC6 selectivity. Notably, the concentration of HDAC1 in the human
brain is 10-fold greater than that of HDAC6,34 therefore, the selectivity of [18F]3 is an important
factor for HDAC6 imaging. Compared with our result, however, a previous study reported a
higher binding affinity of compound 2 for HDAC6, with an IC50 value of 1.40 nM and a 3700
fold selectivity over HDAC1.42 This discrepancy may have been caused by differences in
enzyme activity assay systems. Additionally, it was reported that one of the two catalytic
domains of HDAC6 deacetylated a broad variety of substrates, whereas the other catalytic
domain has a high specificity for substrates with C-terminal acetyllysine residues.13 Therefore,
inhibition assays for HDAC6 activity using fluorogenic substrates specific for one catalytic
domain may overlook interactions between the inhibitors and the other catalytic domain. To
determine the accurate binding affinities of ligands for the HDACs and dispel the above
concerns in the future, the development of direct binding assay system, such as a saturation
binding assay using radiolabeled ligands, is required.
3.5. Biodistribution in normal mice
The biodistribution of [11C]2 and [18F]3 from 1 min to 60 min post-injection was evaluated in
normal mice (Table 2). Excellent stability of [11C]2 was demonstrated in plasma radio-
metabolite analysis in mice;39 therefore, the comparison of pharmacokinetics between the
radioligands could reveal the influence of structural modifications, such as the introduction of
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an [18F]fluoroethyl moiety, of [18F]3 on its pharmacokinetics. The initial blood radioactivity of
[18F]3 was higher than that of [11C]2 (4.3% ID/g versus 2.3% ID/g at 2 min post-injection),
although both radioactivity levels gradually decreased over time and reached the same degree
at 60 min post-injection. Similarly, in some organs the radioactivity uptake of [18F]3-injected
mice was significantly higher than observed with [11C]2-injected mice, including the heart,
lung, liver, and muscle, at the early time points. High radioactivity was observed in the liver,
kidney, and small intestine, suggesting that the radioligands were excreted by both renal and
hepatobiliary excretion. Brain uptake of [11C]2 was less than 0.5% ID/g over a 60-min period,
and this observed poor BBB permeability is consistent with previous reports.38,39 Similarly,
brain uptake of [18F]3 was low and stable at around 0.5% ID/g over a 60-min period. Murine
testis is a known to exhibit high expression levels of HDAC6.47 The development and function
of the testis of HDAC6-knockout mice are normal,48 whereas inhibition of HDAC6 restricts
sperm motility in rats through increased levels of acetyl α-tubulin, which is a component of
microtubules in the flagella.49 In our study, however, testis radioactivity was below blood levels
at every time point for both [11C]2 and [18F]3. As with the brain, the testis has a blood-testis
barrier to protect spermatocytogenesis from toxicants in the systemic circulation and
autoimmune responses;50 therefore, the barrier may inhibit testis uptake of the radioligands.
Although radioactivity uptake in the brain and testis of [18F]3-injected mice was higher than in
[11C]2-injected mice at several time points, the brain-to-blood and testis-to-blood uptake ratios
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of the radioligands were almost identical. Bone radioactivity of [18F]3-injected mice peaked at
2.6% ID/g at 10 min post-injection followed by a slight decrease over time. The [18F]fluoride
ion is characterized by accumulation in the bone, therefore it appears that [18F]3 has a high
tolerance to in vivo defluorination.
The deacetylation and ubiquitin-binding functions of HDAC6 play important roles in cancer
growth, cell survival, and metastasis through many signaling pathways,22 therefore,
noninvasive imaging of HDAC6 will aid diagnosis or development of HDAC6-targeting drugs
for cancers. However, because [18F]3 demonstrated high radioactivity uptake in the organs of
the abdomen and chest, the application of this radioligand will be limited to cancers localized
in other areas. For example, increased expression of HDAC6 is observed in several cancers,
including oral squamous cell carcinoma and malignant melanoma.51,52 The expression of
HDAC6 is regulated by estradiol in estrogen receptor α positive breast cancer cells and may
serve as a prognostic indicator.17,53 Furthermore, HDAC6 selective inhibitors have been
evaluated in pre-clinical and clinical trials against cancers such as glioblastoma, multiple
myeloma, and malignant melanoma.23,54 At the late time points in the biodistribution assay, the
radioactivity levels of blood and muscle of [18F]3-injected mice were comparable to those of
[11C]2-injected mice, which demonstrated specific uptake to B16F10 melanoma cells grafted
in mice with a tumor/muscle ratio of approximately 3 in a small animal PET study.39 Therefore,
use of [18F]3 for PET imaging of these cancers is justified.
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3.6. Metabolite analysis
The metabolic stability of [18F]3 in mouse plasma was determined by HPLC analysis (Figure
3 and Table 3). The percentages of the intact radioligand gradually decreased with time and
were 76 ± 3% and 70 ± 12% at 15 and 30 min post-injection, respectively. In addition to
unchanged [18F]3, two polar radioactive metabolites were observed in mouse plasma. In
comparison, analysis of the metabolism of the Tubastatin A analogue [11C]KB631 indicated
that the percentage of the unchanged form in mouse plasma were 98 and 84% after 10 and 60
min post-injection, respectively.39 The in vivo stability of [11C]Martinostat in rodents and non-
human primates has been reported. The degradation of [11C]Martinostat is limited at 30 min
post-injection in rat brains,33 whereas the proportion of unchanged [11C]Martinostat remained
around 40 % in plasma 30 min post-injection in baboons.33,55 A study using carbon-11 labeled
MS-275, a benzamide HDAC inhibitor undergoing clinical trials for cancer treatment, reported
that the percentage of the unchanged form in baboon plasma was approximately 60% 40 min
post-injection.56 Compared with these radioligands, the metabolic stability and tolerance to
defluorination in mice of [18F]3 are considered to be acceptable for in vivo imaging, but further
studies to characterize the chemical species of radiometabolites and to determine the stability
in non-human primates are needed.
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3.7. Effect of P-gp inhibition on brain uptake of [18F]3
We assessed the influence of P-gp, a multidrug-resistance-associated protein, on the brain
kinetics of [18F]3. Small animal PET studies were performed in mice with or without pre-
treatment with the P-gp inhibitor CsA, and the brain time-activity curves were compared
(Figure 4a and 4b). P-gp inhibition had no effect on the brain uptake of [18F]3 until 10 min after
injection. On the other hand, time-activity curves showed that the brain radioactivity of mice
with P-gp inhibition was slightly higher than that of control mice during the late scan period.
In summed PET images of the mouse heads at 30 min post-injection (Figure 4c), an increase
in back-ground radioactivity was confirmed in the mice with P-gp inhibition compared with
the control mice. This increased radioactivity appeared to spill over into the brain region of
interest, resulting in the observed difference in the time-activity curves. It is known that CsA
is a substrate of cytochrome P450 3A4;57,58 therefore, inhibition of this enzyme by CsA
treatment may have reduced the metabolism and excretion of [18F]3 in mice.
Generally, characteristics such as low molecular weight (<500 Da) and moderate lipophilicity
(2.0–3.5 in log D) indicate, but do not guarantee, that compounds will exhibit good BBB
permeability.46 The molecular weight and log D value of 3 are 367 Da and 2.0, respectively;
however, its brain uptake in mice is quite low. This discrepancy may be attributed to, as
Kozikowski et al. suggested, the effect of pKa values of certain HDAC inhibitors with a
hydroxamic acid moiety on BBB permeability.59 These authors used in vitro membrane
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permeability studies to demonstrate that compounds containing a basic cap group with a high
pKa value (more than approximately 5.9) tended to have low membrane permeability, which
was likely due to ionization at physiological pH. The pKa value of the basic cap group of
Tubastatin A is relatively high (7.14);59 therefore, lowering the pKa values by structural
modification may improve the BBB permeability of Tubastatin A analogues.
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4. CONCLUSIONS
In summary, we report the radiosynthesis and biological evaluation of a novel 18F-labeled
Tubastatin A analogue [18F]3 for PET imaging of HDAC6. The radiosynthesis of [18F]3 was
achieved by a two-step reaction composed of 18F-fluorination and hydroxamic acid formation,
which was readily applied to an automatic radiosynthesizer. The HDAC6 selectivity of 3 was
almost identical to that of a methylated analogue KB631. Biodistribution studies in mice
revealed the poor BBB permeability of [18F]3, suggesting a limitation for studies into
neurodegenerative diseases. Meanwhile, the characteristics of [18F]3, such as steady clearance
from blood and muscle, moderate metabolic stability, and high tolerance to in vivo
defluorination, encourage further evaluation of its utility for imaging of certain cancers.
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ACKNOWLEDGEMENTS
We thank Mr. Kunpei Hayashi, Mr. Masanari Sakai, and Mr. Kosuke Nishino (SHI Accelerator
Service Ltd., Tokyo, Japan) for technical assistance with the cyclotron operation and
radiosynthesis. This work was supported by a Grant-in-Aid for Research Activity Start-up No.
16H07486 and a Grant-in-Aid for Young Scientists No. 18K15655 from the Japan Society for
the Promotion of Science and an internal grant from Tokyo Metropolitan Institute of
Gerontology.
CONFLICT OF INTEREST
Jun Toyohara received a research grant from Sumitomo Heavy Industries (Tokyo, Japan).
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