Novel 64Cu-labeled CUDC-101 for in vivo PET imaging of histone deacetylases

Article abstract of DOI:10.1021/ml400191z

We report the design, synthesis, and biological evaluation of a ⁶⁴Cu-labeled histone deacetylase (HDAC) imaging probe, which was obtained by introduction of metal chelator through click reaction of HDAC inhibitor CUDC-101 and then radiolabeled with ⁶⁴Cu. The resulting ⁶⁴Cu-labeled compound 7 ([⁶⁴Cu]7) was identified as a positron emission tomography (PET) imaging probe to noninvasively visualize HDAC expression in vivo. Cell based competitive assay established the specific binding of [⁶⁴Cu]7 to HDACs. Biodistribution and small-animal microPET/CT studies further showed that [⁶⁴Cu]7 had high tumor to background ratio in the MDA-MB-231 xenograft model, a triple-negative breast cancer with high expression of HDACs. To our knowledge, [⁶⁴Cu]7 thus represents the first ⁶⁴Cu-labeled PET HDAC imaging probe, which exhibits nanomolar range binding affinity and capability to imaging HDAC expression in triple-negative breast cancer in vivo.

Full text of DOI:10.1021/ml400191z

Letter
pubs.acs.org/acsmedchemlett
64
Novel Cu-Labeled CUDC-101 for in Vivo PET Imaging of Histone
Deacetylases
Qingqing Meng,† Feng Li,† Sheng Jiang,‡ and Zheng Li*,†
†Department of Translational Imaging, The Methodist Hospital Research Institute, Houston, Texas 77030, United States
‡Laboratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou
510530, China
S
* Supporting Information
ABSTRACT: We report the design, synthesis, and biological
evaluation of a 64Cu-labeled histone deacetylase (HDAC)
imaging probe, which was obtained by introduction of metal
chelator through click reaction of HDAC inhibitor CUDC-101
and then radiolabeled with 64Cu. The resulting 64Cu-labeled
compound 7 ([64Cu]7) was identified as a positron emission
tomography (PET) imaging probe to noninvasively visualize
HDAC expression in vivo. Cell based competitive assay
established the specific binding of [64Cu]7 to HDACs.
Biodistribution and small-animal microPET/CT studies further showed that [64Cu]7 had high tumor to background ratio in
the MDA-MB-231 xenograft model, a triple-negative breast cancer with high expression of HDACs. To our knowledge, [64Cu]7
thus represents the first 64Cu-labeled PET HDAC imaging probe, which exhibits nanomolar range binding affinity and capability
to imaging HDAC expression in triple-negative breast cancer in vivo.
KEYWORDS: PET imaging probe, 64Cu radiolabeling, histone deacetylases inhibitors, CUDC-101, triple-negative breast cancer
istone deacetylases (HDACs) are a class of enzymes
biological and molecular events of HDACs, there is an
Hmodulating gene expression via deacetylation of both increasing need for HDAC targeting imaging probes. To
date, limited noninvasive HDAC imaging probes were reported
for cancer diagnosis and therapy evaluation of HDACi in
vivo.13 For example, in Figure 1, [18F]fluoroacetamide-1-
hexanoicanilide (FAHA) and [11C]MS-275 were reported for
brain cerebral HDAC imaging.14,15 18F-suberoylanilide hydroxa-
mic acid (18F-SAHA) was reported to be able to evaluate the
target efficacy of HDAC inhibitor SAHA within 24 h of drug
administration in a murine ovarian cancer model.16
Breast cancer is one of the leading causes of cancer death in
women. Recent studies found that HDACs were correlated
with estrogen receptor (ER) and progesterone receptor (PR)
expression, and its expression enabled a more precise
assessment of the prognosis of breast cancer patients.17,18
Introducing HDACi in breast cancer treatment could overcome
resistance to hormonal therapy.19 HDACi was also reported to
selectively target triple-negative breast cancer cell proliferation
and survival in vitro and tumorigenesis in vivo.20 HDACs as a
promising biomarker play an increasingly important role in
breast cancer treatment. For breast cancer imaging, targeted ER
and human epidermal growth factor receptor 2 (HER2)
imaging probe is currently under intense preclinical and clinical
investigation; however, these agents only apply to ER or HER2-
positive subtypes.21 Imaging probes targeting other major
histone and nonhistone proteins, and thereby, these enzymes
are involved in a wide range of biological processes.1−3 Many
HDAC inhibitors have shown excellent properties for tumor
therapy in clinical trials. To date, 18 mammalian HDAC
subtypes have been discovered and accordingly classified into
four classes based upon their phylogeny.4,5 Class I (HDACs 1−
3 and 8), class IIa HDACs (4, 5, 7, and 9), class IIb HDACs (6
and 10), and the class IV (11) are Zn-dependent hydrolases,
whereas class III (sirtuins 1−7) enzymes are NAD+-dependent
Sir2-like deacetylases.5 Class I HDACs are mostly localized
within the nucleus, whereas class II HDACs shuttle between
nucleus and cytoplasm. HDAC isoforms are highly expressed in
various cancers, including breast cancer, pancreatic cancer,
colorectal cancer, prostate cancer, and hepatocellular carcino-
ma.6−8 Treatment of tumor cells with HDAC inhibitors results
in growth arrest, differentiation, and apoptosis, promoting these
enzymes as potential cancer drug targets.9,10
HDAC inhibitors (HDACi) are divided into several
structural classes including hydroxamates, cyclic peptides,
aliphatic acids, and benzamides.11 So far, more than 11 new
HDACi are in various stages of clinical development for therapy
of multiple cancer types.12 Among them, suberoylanilide
hydroxamic acid (SAHA, Vorinostat, Merck Research Labo-
ratories) and FK228 (Romidepsin, Celgene)2 have been
approved by US FDA for the treatment of cutaneous T-cell
lymphoma. To evaluate the efficiency of existing and novel
HDACi and acquire important basic scientific insight of
Received: May 17, 2013
Accepted: July 25, 2013
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Radiosynthesis of [64Cu]7
a
Reagents and conditions: (i) 6-azidohexan-1-amine, sodium
ascorbate, CuSO4, H2O/t-BuOH, r.t., 24 h. (ii) DOTA−NHS, NEt3,
DMSO. (iii) 64CuCl2, 0.1 M NH4OAc (pH = 5.5), 60 °C, 1 h.
Figure 1. Chemical structures of representative HDAC inhibitors and
HDAC PET imaging probes.
cellular receptors or biomarkers like HDACs are highly in
demand for breast cancer diagnosis and therapy evaluation
especially for triple-negative subtype patients who usually have
poor prognosis. Herein, to our best of knowledge, we report the
first 64Cu-labeled (t1/2 = 12.7 h; β+ 655 keV, 17.8%) HDAC
PET imaging probe [64Cu]7 developed from a HDACi, 7-(4-
(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hy-
droxyheptanamide (CUDC-101, Selleckchem, TX), which is
currently in phase I clinical trials with multiple tumor types
including advanced breast cancer.22,23
As a member of hydroxamates HDACi, CUDC-101 has the
distinguishing characteristics including a hydroxamic acid group
interacting with zinc at the active site of HDACs to interfere
with enzyme activity, carbon linker, and capping group.24 The
capping group is solvent-exposed and interacts with amino
acids near the entrance of the active site.25,26 In this study,
chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
(DOTA, Macrocyclics, TX) was conjugated to CUDC-101 with
a hydrophobic carbon chain linker by click reaction. Our
synthesis started from commercially available CUDC-101,
which was then reacted with a carbon chain linker bearing an
azide group at one end as well as a primary amine group at the
other end. After the introduction of the linker, compound 9 was
conjugated to DOTA-NHS in the presence of NEt3 to give
compound 7 (Scheme 1) (Supporting Information).
3.0 GBq/mg (2.4−2.9 MBq/nmol) at the end of synthesis.
[64Cu]7 was stable in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (FBS) for up to
24 h at 37 °C (Supporting Information).
To determine the HDAC specificity of [64Cu]7, a
competitive cell binding assay was performed by competition
of [64Cu]7 with increasing concentration of HDAC inhibitor
CUDC-101 using MDA-MB-231, a triple-negative human
breast cancer cell line with high HDACs expression.27 The
cellular retention and internalization of radioactivity was
determined using a γ counter (Perkin-Elmer Packard, CT).
The results showed that the cell uptake of [64Cu]7 decreased
along with the increasing of the CUDC-101 concentration,
which provided evidence for target-mediated binding to
HDAC-expressing cells (Figure 2). This competitive cell
binding assay that established the cell uptake of [64Cu]7 was
realized by its specific binding to HDAC in vitro.
The in vivo imaging of mice bearing human triple-negative
breast cancer MDA-MB-231 xenograft tumors was carried out
We subsequently determined HDAC binding activity of
compound 7 using the Fluor-de-Lys HDAC Fluorimetric
Assay/Drug Discovery Kit (Enzo Life Sciences, NY). The IC50
value of 7 was measured as 94.47
19.24 nM (Supporting
Information Figure S1). This finding thus identified 7 as a
promising candidate for the development of 64Cu HDAC
targeting PET imaging probe. Compound 7 was radiolabeled
by incubating with 64CuCl2 (Washington University, MO) in
0.1 N NaOAc buffer (pH 5.5) at 60 °C for 1 h (Scheme 1).
The labeled product [64Cu]7 was then directly purified by
radio-HPLC with ≥98% radiochemical yield and chemical
purity. The specific activity of [64Cu]7 was estimated to 2.5−
Figure 2. In vitro dose−response profile for [64Cu]7 competition with
CUDC-101 on the MDA-MB-231 breast cancer cells.
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ACS Medicinal Chemistry Letters
Letter
by static microPET/CT scans at 2, 6, and 24 h after tail-vein
injection of [64Cu]7 (100 μCi, 3.7 MBq) (n = 4).
Representative coronal slices with tumor are shown in Figure
3a. The radioactive uptake in the PET images was expressed as
was measured using γ-counter (Perkin-Elmer Packard, CT).
The decay-corrected %ID/g of organs and tumors are shown in
Table 1.
Table 1. Decay-Corrected Biodistribution of [64Cu]7 with or
without Co-Injection of CUDC-101 in MDA-MB-231
a
Tumor Bearing Mice 24 h after Injection (n = 3)
tissue
blood
[64Cu]7
[64Cu]7 + CUDC-101
0.50 0.07
1.07 0.01
1.51 0.14
3.23 1.25
0.88 0.35
1.90 0.06
2.21 0.18
0.23 0.02
0.75 0.40
0.56 0.26
1.09 0.17
1.54 0.41
2.51 0.53
0.99 0.14
1.69 0.32
1.36 0.23
0.24 0.06
0.84 0.26
heart
lung
liver
spleen
kidney
tumor
muscle
GI
tumor-to-normal tissue uptake ratio
tumor/muscle
tumor/blood
9.61 1.54
4.44 0.88
5.70 0.48
2.64 0.83
a
The data percentage of the injected dose per gram of tissue (%ID/g)
except tumor-to-muscle ratio. Values are quoted as mean standard
deviation (SD).
The biodistribution result demonstrated that the tumor
uptake of [64Cu]7 reached 2.21 0.19%ID/g at 24 h p.i. in the
imaging group, whereas the presence of nonradioactive CUDC-
101 significantly reduced the tumor uptake to 1.36
0.23%
ID/g (P < 0.01) in the blocking group with a 38% decrease. For
the imaging group (nonblocking), 3.23 1.25% ID/g and 1.90
0.06% ID/g remained in the liver and kidneys, respectively.
The overall uptake of [64Cu]7 in other tissues and organs of
blocking group was similar to that of the nonblocking group.
On the basis of the biodistribution results, the contrast ratio of
tumor to muscle for the imaging groups was calculated as 9.61
1.53, which made tumors clearly visible at 24 hs p.i. as shown
in Figure 3a. Meanwhile, the corresponding value for the
blocking group was 5.70 0.48 with a 38% decrease. These
data proved the HDAC specific binding of [64Cu]7 in vivo. The
biodistribution results were consistent with the quantitative
analysis of microPET imaging.
Figure 3. (a) Decay-corrected microPET/CT scan of MDA-MB-231
tumor bearing mice (n = 4) at 2, 4, and 24 h after i.v. injection of
[64Cu]7. The image obtained with coinjection of CUDC-101 (20 mg/
kg body weight) is shown for a 24 h blockade (right). Tumors are
indicated by arrows. (b) Decay-corrected region-of interest (ROI)
analysis on microPET images of the tumor uptake of [64Cu]7 with or
without coinjection of CUDC-101 (20 mg/kg body weight). *, P <
0.05; **, P < 0.01.
a percentage of the injected radioactive dose per gram of tissue
(%ID/g). From the PET imaging, the tumor was clearly visible
as early as 2 h postinjection (p.i.). [64Cu]7 had the high uptake
in the liver, kidney, and tumor, with relatively low distribution
in other tissues at 24 h p.i. Region-of-interest (ROI) analysis on
microPET images showed that the tumor uptake values were
1.20 0.21, 2.16 0.08, and 2.36 0.31% ID/g at 2, 6, and 24
h p.i., respectively (Figure 3b). To test the HDAC specificity of
[64Cu]7 in vivo, blocking experiments were performed by
coinjection of 20 mg/kg nonradioactive HDAC inhibitor
CUDC-101 with [64Cu]7 . The MDA-MB-231 tumor uptake
values for blocking experiments were 0.48 0.24, 0.94 0.37,
and 1.22 0.31% ID/g at 2, 6, and 24 h p.i., respectively, which
showed significant decreasing compared with that of the
imaging group at all time points (Figure 3b). In the blood
clearance study shown in Figure S2 (Supporting Information),
[64Cu]7 was rapidly cleared from the blood. The rapid
clearance continued until 30 min postinjection when only 6%
ID/g was left in the blood.
In conclusion, we reported, to our knowledge, the first 64Cu
labeled probe [64Cu]7 for PET imaging of HDACs in vivo
using a triple-negative breast cancer xenograph model. From
the enzymatic assay, the IC50 of this compound to HDACs was
shown to be in the nanomolar range, which indicated the
remaining HDAC activity during structure modification from
HDAC inhibitor CUDC-101. The specific binding of radio-
labeled probe [64Cu]7 to HDACs was proven by the
competition of [64Cu]7 with increasing concentration of
CUDC-101 using MDA-MB-231 breast cancer cells. Micro-
PET/CT imaging revealed rapid and high [64Cu]7 uptake in
MDA-MB-231 breast tumors, and HDAC specificity in vivo was
also confirmed by blocking experiments. This proof-of-concept
research demonstrated the feasibility of using small molecule
HDAC inhibitor for HDAC imaging probe development.
Further optimization of [64Cu]7 by using different Cu chelators
to achieve better distribution and tumor uptake will be
performed in a following study. The success of HDAC specific
imaging in breast cancer using [64Cu]7 has potential to be
translated in clinical applications to noninvasively document
We subsequently determined the tissue distribution of
[64Cu]7 by sacrificing the mice at 24 h p.i. immediately after
microPET/CT scans. Tumor, organs, and tissues of interest
were excised and weighed; the radioactivity of harvested tissues
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ASSOCIATED CONTENT
* Supporting Information
■
S
Full experimental details for compounds synthesized, proce-
dures for radiolabeling, description of enzymatic and cell based
assays, biodistribution and small animal PET imaging studies.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(Z.L.) Tel: +1 713-441-7962. Fax: +1 713-790-3018. E-mail:
zli@tmhs.org.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This research was funded (or supported) by The Methodist
Hospital Research Institute.
Notes
The authors declare no competing financial interest.
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ABBREVIATIONS
■
HDAC, histone deacetylase; HDACi, histone deacetylase
inhibitor; SAHA, suberoylanilide hydroxamic acid; ER, estrogen
receptor; HER2, human epidermal growth factor receptor 2;
PET, positron emission tomography; FAHA, [18F]-
fluoroacetamide)-1-hexanoicanilide; DOTA, 1,4,7,10-tetraaza-
cyclododecane-1,4,7,10-tetraacetic acid; ROI, region-of-interest
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Mita, K.; Hamaguchi, M.; Hara, Y.; Kobayashi, S.; Iwase, H.
Quantitation of HDAC1 mRNA expression in invasive carcinoma of
the breast. Breast Cancer Res. Treat 2005, 94 (1), 11−16.
(19) Thurn, K. T.; Thomas, S.; Moore, A.; Munster, P. N. Rational
therapeutic combinations with histone deacetylase inhibitors for the
treatment of cancer. Future Oncol. 2011, 7 (2), 263−283.
(20) Tate, C. R.; Rhodes, L. V.; Segar, H. C.; Driver, J. L.; Pounder,
F. N.; Burow, M. E.; Collins-Burow, B. M. Targeting triple-negative
breast cancer cells with the histone deacetylase inhibitor panobinostat.
Breast Cancer Res. 2012, 14 (3), R79.
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