Acetazolamide-based [18F]-PET tracer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors

Article abstract of DOI:10.1016/j.bmcl.2018.01.060

Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized novel [¹⁸F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models other than SKRC-52. [¹⁸F]-acetazolamide (1) was found to be insufficient for the specific accumulation in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Full text of DOI:10.1016/j.bmcl.2018.01.060

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acetazolamide-based [18F]-PET tracer: In vivo validation of carbonic
anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic
solid tumors
Kunal N. More a,1, Jun Young Lee b,1, Dong-Yeon Kim c, Nam-Chul Cho d, Ayoung Pyo c, Misun Yun c,
Hyeon Sik Kim c, Hangun Kim a, Kwangseok Ko d, Jeong-Hoon Park b, , Dong-Jo Chang a,
⇑
⇑
a College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea
b Radiation Instrumentation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea
c Department of Nuclear Medicine, Chonam National University, Hwasun Hospital, Hwasun 58128, Republic of Korea
d C&C Research Laboratories, DRC, Sungyunkwan University, Suwon 16419, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential
target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in
clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of
CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized
novel [18F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and
performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models
other than SKRC-52. [18F]-acetazolamide (1) was found to be insufficient for the specific accumulation
in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide
PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in
future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX
is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.
Received 12 September 2017
Revised 25 January 2018
Accepted 28 January 2018
Available online xxxx
Keywords:
Carbonic anhydrase IX
Acetazolamide
Hypoxic tumor
PET imaging
Target validation
Ó 2018 Elsevier Ltd. All rights reserved.
Tumor hypoxia is a salient feature of broad numbers of solid
tumors including frequent observations in distant metastasis.
Hypoxic tumors with low level of O2 below 20 mmHg are more
prone to aggressive nature with poor prognosis, resistance to can-
cer therapy and low survival rate of cancer patients.1,2 Moreover,
hypoxic cells are more resistant to radiation and chemotherapy
than normoxic cells.3 Thus, hypoxia becomes a sign of advanced
stage tumor; detection of which might be helpful to locate the
tumor site. Recent discovery suggested many biomarkers for detec-
tion of hypoxic tumors microenvironment such as HIF-1 for cellu-
lar processes, vascular endothelial growth factor (VEGF) for
angiogenesis, carbonic anhydrase IX (CA-IX) for pH regulation,
and glucose transporter 1 (GLUT-1) for metabolism.4
tumor hypoxia.2,5 Hypoxia-induced stabilization of HIF-1
a causes
the expression of gene for CA-IX enzyme which alters cell home-
ostasis and consequently induces various oncogenic processes such
as cell proliferation, angiogenesis, cell invasion, metastasis and
metabolic changes in hypoxic tumors.6,7 Among 15 human iso-
zymes of carbonic anhydrase (CA), CA-IX is a most active CA for
the CO2 hydration reaction and prominently overexpressed in
hypoxic tumors.8 Overexpression of CA-IX plays a pivotal role in
many cancers as non-small cell lung cancer (NSCLC),9 breast,10,11
renal cancer,12 ovarian,13 brain,14 colon, and head and neck
cancers.13,15 Expression of CA-IX in different cancers with featured
hypoxia and presence of extracellular active site make it a promis-
ing target for anticancer therapy as well as an attractive target for
drug delivery purpose such as imaging and therapy.16 The crystal
structure of CA-IX enables to develop potent and selective small
molecule inhibitors targeting CA-IX. Structurally, carbonic anhy-
drase is a metalloenzyme, and the active site is formed as distorted
tetrahedral geometry with zinc ion as center in coordination with
three imidazole groups of histidines and one hydroxide ion of sub-
strate.17 Recent paradigm in cancer therapeutics is the develop-
ment of new CA-IX inhibitors for better efficacy in alone or in
CA-IX is a highly active cell surface anchored enzyme involved
in hypoxia-induced stress, and is regulated by transcription factor,
hypoxia-inducible factor-1a (HIF-1a) activated in response to
⇑
Corresponding authors.
E-mail addresses: parkjh@kaeri.re.kr (J.-H. Park), djchang@sunchon.ac.kr
(D.-J. Chang).
1
The first two authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2018.01.060
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: More K.N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.060

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K.N. More et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
combination with chemotherapeutic agent or radiotherapy.7,18,19
Small molecules inhibitors targeting CA-IX are either coumarin sui-
cidal inhibitors or sulfonamides moiety.13,20,21 Sulfonamide ligand
with high affinity to central metal ion of an enzyme can be utilized
for the targeted delivery of potent cytotoxic drugs into solid
tumors, which is otherwise showed ineffective biodistribution to
desired tumor site and accumulated in normal tissues.19,22 Devel-
opment of CA-IX inhibitors as an anticancer agent also enhanced
the development of PET imaging ligand for diagnostics and small
molecule-drug conjugates (SMDCs) for therapeutic purpose in both
solid and metastatic tumors. Acetazolamide, a prototype23 of sul-
fonamides, is a clinically approved pan-CA inhibitor with good pro-
file,21,24 and is also known to possess antitumor activity alone or in
combination.18,25 Suppression of tumor metastasis in lung carci-
noma was also acquired by acetazolamide.26 99mTc-labeled aceta-
zolamide was even used as a ligand for development of PET
imaging radiotracer for imaging purpose27 and SMDCs with aceta-
zolamide ligand for pharmacodelivery of cytotoxic drug at tumor
site.28 However, most of the known radiotracers for imaging CA-
IX as 99mTc-labeled acetazolamide ligand, 124I-cG250 or 124I- and
89Zr-labeled antiCA-IX monoclonal antibodies were performed
well predominantly for in vivo imaging of CA-IX in hypoxia inde-
pendent renal cell carcinoma SKRC-52,27,29 even though CA-IX is
also expressed in various hypoxic tumor models.30 Only few PET
tracer as 68Ga-NOTGA-(AEBSA)3 and 18F-AmBF3-(ABS)3 with tri-
meric sulfonamide moieties achieved the proposed role. Surpris-
ingly, the tracer with trimeric sulfonamide moieties showed
higher uptake with selective targeting to CA-IX compared to mono-
meric and dimeric isoforms.22 Reported studies raised a question
that CA-IX is really a universal target for imaging of all solid tumors
other than SKRC-52. One review was published performing valida-
tion of CA-IX target for hypoxic tumor imaging.31 The review pro-
posed that CA-IX is an unreliable target for hypoxic imaging due to
the facts that CA-IX expression is cancer type-dependent, not all
hypoxic tumors express CA-IX, expression of CA-IX is undetectable
in certain cancer types, and re-oxygenation of previously hypoxic
cancer cells induces expression of CA-IX. However, no in vivo
experimental study was carried out to support this conclusion
yet. Thus, further validation of CA-IX as a target for development
of clinically feasible hypoxia imaging PET tracer is required to be
performed. In the view of validation of CA-IX target for hypoxic
tumor imaging, we designed and synthesized a new [18F]-PET tra-
cer (1) based on acetazolamide as shown in Scheme 1. In vivo PET
imaging and biodistribution studies of [18F]-PET tracer (1) were
performed in CA-IX positive 4T1 and HT-29 skin xenograft Balb/c
mice tumor models. 4T1 and HT-29 cancer cells are known to
express CA-IX within hypoxic microenvironment. Imaging of PET
tracer was also performed in CA-IX expressed lung metastatic
tumors model formed by 4T1 breast cell lines.11 The in vivo PET
images and biodistribution profile provided insight about the
behavior of our novel PET tracer [18F]-acetazolamide (1) on tumor
model.
The precursor 6 for [18F]-labeling was synthesized to couple
with [18F]-PEG-alkyne (9) to form central triazole ring by copper
(I)-catalyzed click chemistry as shown in Scheme 1. The precursor
6 was synthesized starting with hydrolysis of commercially avail-
able acetazolamide (2) giving compound 3. Compound 3 was
reacted with succinic anhydride in DMF with heating to obtain car-
boxylic acid (4). Compound 4 was subjected to amide coupling
reaction with azide-PEG linker (2-(2-(2-azidoethoxy)ethoxy)
ethan-1-amine) (5) using BOP which finally provided the precursor
(6) for click reaction. This precursor was then used for synthesis of
radiolabeled [18F]-acetazolamide (1) through the click reaction
with [18F]-PEG-alkyne (9). Radiolabeling of the mesylated PEG-
alkyne linker (7) successfully provided [18F]-PEG-alkyne precursor
(9) in 32.5% radiochemical yield with >99% of radiochemical purity.
The click reaction of precursor (6) with [18F]-PEG-alkyne (9) was
performed to obtain final [18F]-PET tracer (1) in radiochemical
yield of 93.73% and radiochemical purity >99% after HPLC purifica-
tion (see Fig. S1 and Table S1, ESIy).
Scheme 1. Reaction conditions and reagents: A. (a) conc. HCl, EtOH, reflux, quant. yield; (b) succinic anhydride, DMF, 100 °C, 96%; (c) 2-(2-(2-azidoethoxy)ethoxy)ethan-1-
amine (5), BOP, iPrNEt2, DMF, rt, 53%; B. (d) TBAF, THF, 80 °C, 33%; (e) 18F/[2,2,2]-cryptand, CH3CN, 100 or 80 °C, 33%; C. (f) CuI, iPrNEt2, CH3CN, rt, 94%.
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K.N. More et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
PET imaging was performed after tail i.v. injection of [18F]-
acetazolamide (1) inside CA-IX expressing 4T1 and HT-29 skin
xenograft tumor models to investigate the efficiency of PET tracer
for its uptake to tumor. (Fig. 1A and B) In vivo PET imaging scans
were performed over different time interval such as 5, 15, 30, 60
and 90 min. Initially, the strong signal was detected in abdomen,
intensity of which was decreased over time at final scans. Micro-
PET imaging study of [18F]-acetazolamide (1) showed undetectable
uptake in 4T1 and HT-29 xenograft tumors against background tis-
sues. This PET imaging study was performed at different time
points p.i. to see the variation in uptake level of [18F]-acetazo-
lamide (1) into tumors over the time. Unfortunately, not a single
time point shows any detectable amount in tumor, suggesting that
PET tracer (1) is not specifically accumulated in CA-IX expressing
tumor.
In order to understand the fate of [18F]-acetazolamide (1) inside
the CA-IX expressed mice body, biodistribution study was per-
formed after harvesting various body organs. (Fig. 2A and B) In this
study, the mouse blood sample was obtained by heart puncture
and then heart, lung, liver, spleen, stomach, intestine, pancreas,
kidney, muscle, fat, bone, skin, tail, tumor, and brain were har-
vested, weighed and counted by using gamma counter at each time
interval at 15, 30, 60 and 90 min. [18F]-Acetazolamide (1) was
localized in tumor with very trace amount but most is accumu-
lated in intestine in both cases of tumor types. Kidney and stomach
were also shown detectable signal which diminuend over the time.
Tumor model with HT-29 cells, when compared to 4T1 xenograft,
retained more amount of PET in blood compared to tumor. The
high blood uptake in HT-29 might be due to binding to CA-II in
red blood cell. In case of 4T1 xenograft, intestinal amount of
Fig. 1. A. MicroPET images of Balb/C mice xenograft bearing 4T1 cells at 5, 15, 30, 60 and 90 min. B. Coronal microPET images (top) and maximum intensity projection
(bottom) microPET images of Balb/C mice bearing HT-29 cells at 5, 15, 30, 60 and 90 min. Tumor positions are indicated by white arrows.
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K.N. More et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 2. Biodistribution study of [18F]-acetazolamide (1). A. On Balb/C xenograft model bearing 4T1 cells at 15, 30, 60 and 90 min. B. On Balb/C xenograft model bearing HT-29
cells at 15, 30, 60, and 90 min.
[
18F]-acetazolamide (1) decreased over time showing fast clear-
metastases. The imaging scan showed no accumulation of PET tra-
cer in metastatic lung tumor, with more accumulation in abdomen
like xenograft tumor model even after 120 min.
ance rate from intestine, whereas in HT-29 most amount accumu-
lated in intestine for longer time, and only trace amount of PET
tracer was passed to tumor. Tumor to blood and tumor to muscle
ratio of tracer in 4T1 xenograft didn’t show any change over time
suggesting inefficient uptake in tumor. It was confirmed that
The pharmacokinetic nature of [18F]-acetazolamide (1) was
assessed through the evaluation of partition coefficient, in vitro sta-
bility in human serum and in vivo stability in Balb/C mice. The log p
value of ꢀ1.87 0.0093 suggested the hydrophilic nature of [18F]-
acetazolamide (1) (see Table S1, ESIy). Hydrophilic tracers have
some advantages as less prone to blood protein binding allowing
high fraction of a free drug, which leads the drug permeation to
reach tissues, and faster clearance kinetics with low toxicity. As
shown in Fig. 4, [18F]-acetazolamide (1) was found to be almost
81% intact in human serum after 90 min (also see Table S2, ESIy).
In vivo stability study of [18F]-Acetazolamide in Balb/C mice
showed that 50% of the PET tracer was intact after 30 min with
decreasing stability with 20% at 1hr and 10% after 2hr post injec-
tion (see Table S3, ESIy). The low signal in bone (Fig. 2) suggested
that the decreased stability of [18F]-acetazolamide (1) is due to
the metabolic degradation by a process other than defluorination
of [18F] fluoride.33 However, the low tumor uptake in PET imaging
[
18F]-acetazolamide (1) was accumulated highly in intestine in
HT-29 and 4T1 xenografts and no signal was found in both tumor
models, which concluded that [18F]-acetazolamide (1) is not tar-
geted well to CA-IX tumor.
CA-IX is a vital biomarker for tumor progression and metastatic
tumors, and hypoxia-dependent CA-IX expression has been known
to induce the metastasis in progress of cancer.11 Metastasis tumors
are more challenging to manage clinically,32 thus CA-IX imaging
would be a great importance in metastatic tumors. To evaluate fea-
sibility of CA-IX imaging in metastatic tumor, we performed the
microPET study of [18F]-acetazolamide tracer (1) in lung metastatic
tumor models induced by 4T1 breast cancer cells as shown in
Fig. 3. Intravenous injection of [18F]-acetazolamide (1) in lung
metastatic tumor model was not successful for detection of lung
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K.N. More et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Fig. 3. MicroPET images of [18F]-acetazolamide (1) in lung metastatic tumor model induced by 4T1 cells at time interval of 30 min, 60 min and 120 min. Tumor positions are
indicated by white arrows.
A
B
100
80
60
40
20
0
0
20
40
60
80
100
120
Time (min)
Fig. 4. Stability of [18F]-acetazolamide (1). A. In vitro stability in human serum. B. In vivo stability in 8 weeks old Balb/C mice.
does not come from the result of in vitro and in vivo instability
the similar binding affinity and can be a good ligand of CA-IX for
because almost 80% (90 min, in vitro) and 50% (30 min, in vivo) of
PET tracer was intact for significant time span for imaging study,
suggesting [18F]-acetazolamide (1) is a feasible PET tracer for
in vivo imaging.
imaging study.
Adams et al. reported that only 35% of human invasive breast
cancers expressed CA-IX and not a single biomarker target serve
as a sole target for imaging purpose.37 Reason of inefficiency of
Molecular docking study was performed to investigate whether
[F]-acetazolamide (1) could bind to CA-IX. Binding parameters of
our PET tracer were calculated using Schrodinger Suite program
(Schrödinger LLC, NY, USA). The human CA-IX mimic co-crystal
structure (PDB ID: 4K0S) with acetazolamide was used as a model
structure for this docking study. The human CA-IX mimic, which is
a CA-IX analogue engineered by site-specific mutations in the
active site of CA-II, has been powerfully used for the development
of carbonic anhydrase inhibitors.34 Prior to the docking study of
[F]-acetazolamide (1), the binding mode of acetazolamide was first
predicted to confirm the accuracy of docking study by this model-
ing module. As shown in Fig. 5B, the binding mode of acetazo-
lamide in docking study is the almost same as that of the X-ray
co-crystal structure. Based on this result for acetazolamide, the
docking study of [F]-acetazolamide (1) in the hCA-IX mimic crystal
structure was conducted to investigate whether [F]-acetazolamide
(1) has a similar binding mode to acetazolamide. As presented in
Fig 5C and D, the predicted binding mode of [F]-acetazolamide
(1) with the lowest binding scores (Gscore35 ꢁ ꢀ7.96 kcal/mol
and Emodel36 ꢁ ꢀ101.38 kJ/mol), which were similar to those of
acetazolamide (Gscore ꢁ ꢀ9.04 kcal/mol and Emodel ꢁ ꢀ79.19
kJ/mol), showed a good match for the binding conformation of
co-crystalized acetazolamide. This precise analysis of the docking
study suggested that [F]-acetazolamide (1) would probably exhibit
[
18F]-acetazolamide (1) to detect the 4T1 breast cancer and its
metastatic tumor need to be evaluated by additional study. HT-
29 is a popular tumor model for development of CA-IX targeting
tracer. But most of PET tracers are unsuccessful to accumulate in
these tumors except tracers with trimeric structure to achieve
strong affinity for CA-IX.22 No uptake of our synthesized probe
[
18F]-acetazolamide (1) inside tumor in HT-29 xenograft model
motivated to perform a separate detailed study. In conclusion,
we successfully synthesized a novel [18F] radiolabeled acetazo-
lamide ligand and evaluated its potential as CA-IX targeted PET tra-
cer. However, its uptake in both 4T1 and HT-29 tumors was
minimal. The in vivo imaging study of [18F]-acetazolamide (1) in
this context showed that the synthesized acetazolamide-based
PET tracer showed poor uptake in CA-IX expressed hypoxic tumor
on skin xenograft and metastatic xenograft in vivo models. As men-
tioned earlier, CA-IX is overexpressed in many types of cancer, but
most of imaging agents targeting CA-IX are mainly effective on
in vivo model with specific cell lines such as SKRC-52 cells. This
study and previous reports witnessed that CA-IX might not be reli-
able as sole universal target for imaging of CA-IX expressed
hypoxic solid tumors although there was an exception with tri-
meric binding moiety for strong binding. Thus, an additional study
is needed to confirm if the CA-IX is a good target for imaging of
hypoxic tumor or our synthesized PET tracer was unable to
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K.N. More et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 5. Docking study of acetazolamide and [F]-acetazolamide (1) in the active site of human CA-IX mimic crystal structure (PDB ID 4K0S), A. X-ray co-crystal structure of
acetazolamide, B. The predicted binding mode of acetazolamide, C. The predicted binding mode of [F]-acetazolamide (1), D. Superimposition of the predicted conformation of
[F]-acetazolamide (1) with the co-crystallized acetazolamide, E. Binding parameters of acetazolamide and [F]-acetazolamide (1) in docking study. Molecular images were
generated by PyMOL program. (Zn atom: violet sphere, Hydrogen bonds: green dot lines, the interactions with Zn: blue dot lines).
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K.N. More et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
7
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Please cite this article in press as: More K.N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.060