Synthesis and preliminary evaluation of radiolabeled bis(zinc(II)- dipicolylamine) coordination complexes as cell death imaging agents

Article abstract of DOI:10.1016/j.bmc.2011.04.029

The aim of this study was the development of ^99mTc labeled bis(zinc(II)-dipicolylamine) (Zn²⁺-DPA) coordination complexes, and the in vivo evaluation of their usefulness as radiotracers for the detection of cell death. DPA ligand 1 w

Full text of DOI:10.1016/j.bmc.2011.04.029

Bioorganic & Medicinal Chemistry 19 (2011) 3425–3433
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and preliminary evaluation of radiolabeled bis(zinc(II)–dipicolyl-
amine) coordination complexes as cell death imaging agents
Leonie wyffels a, , Brian D. Gray b, Christy Barber a, James M. Woolfenden a, Koon Y. Pak b, Zhonglin Liu a,
⇑
a Department of Radiology, University of Arizona, Tucson, AZ, USA
b Molecular Targeting Technologies, Inc., West Chester, PA, USA
a r t i c l e i n f o
a b s t r a c t
The aim of this study was the development of 99mTc labeled bis(zinc(II)–dipicolylamine) (Zn2+–DPA) coor-
dination complexes, and the in vivo evaluation of their usefulness as radiotracers for the detection of cell
death. DPA ligand 1 was labeled with 99mTc via the 99mTc-tricarbonyl core ([99mTc(CO)3-1]3+) or via HYNIC
(
[
Article history:
Received 31 January 2011
Revised 6 April 2011
Accepted 13 April 2011
Available online 28 April 2011
99mTc-HYNIC-1) in good radiochemical yields. Highest in vitro stabilities were demonstrated for
99mTc(CO)3-1]3+. A mouse model of hepatic apoptosis (anti-Fas mAb) was used to demonstrate binding
to apoptotic cells. 99mTc-HYNIC-1 showed the best targeting of apoptotic hepatic tissue with a 2.2 times
higher liver uptake in anti-Fas treated mice as compared to healthy animals. A rat model of ischemia-
reperfusion injury was used to further explore the ability of the 99mTc-labeled Zn2+–DPA coordination
complexes to target cell death. Selective accumulation could be detected for both tracers in the area at
risk, correlating with histological proof of cell death. Area at risk to normal tissue uptake ratios were
3.82 for [99mTc(CO)3-1]3+ and 5.45 for 99mTc-HYNIC-1.
Keywords:
Cell death
99mTc
Molecular imaging
Zn2+–DPA
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
cells, PS is maintained almost exclusively in the inner monolayer
of the cell membrane. A hallmark of apoptosis is the externaliza-
Cell death can result from two different but closely related pro-
cesses, apoptosis and necrosis. Apoptosis or programmed cell death
plays a key role not only in normal tissue homeostasis but also in
the etiology and pathology of a plethora of diseases. Pathologies
associated with too much apoptosis include AIDS,1 organ transplant
rejection,2 neurodegenerative disorders such as Alzheimer’s dis-
ease,3 and cardiovascular diseases such as ischemia-reperfusion
injury.4 Too little apoptosis, on the other hand, can lead to the
development of cancer.5 Besides its participation in disease, apop-
tosis may also play an important role in disease treatment. Anti-
cancer therapies including chemotherapy and radiation therapy
function by promoting apoptosis in the target cells.6 Necrosis, on
the other hand, results from physiological damage and sudden met-
abolic failure.7 It is a disorganized, passive form of cell death, char-
acterized by swelling and rupture of the cell membrane, resulting in
activation of an inflammatory response.
tion of phosphatidylserine (PS) to the outer side of the plasma
membrane.8 When cells become apoptotic, translocase and flop-
pase that normally keep PS inwards become deactivated while a
third enzyme called scramblase is activated.9 This results in a
redistribution of phospholipids across the bilayer and consequent
externalization of PS to the outer leaflet of the cell membrane.
The appearance of PS on the cell membrane surface is an early sign
that the cell death program has been activated,10 and serves as a
signal for phagocytosis and removal.11 Since PS is an anionic
phospholipid, the externalization alters the cell surface charge to
negative.11,12 In necrotic cells, PS becomes accessible due to pas-
sive rupture of the cell membrane. The presence of PS on the cell
surface provides an abundant molecular marker for detection of
apoptotic and necrotic cells.
The most well known tracers for the detection of apoptosis
based on targeting of externalized PS are based on annexin V. An-
nexin V is a member of the annexin protein family and is known to
bind with high affinity to PS in a Ca2+ dependent manner.13,14
Consequently, annexin V conjugates have been developed for
in vitro and in vivo detection of apoptosis and necrosis.7 Several
radiolabeled derivatives of annexin V have been developed for
PET (18F, 68Ga, 124I),15–17 and SPECT (99mTc, 123I)18,19 imaging of cell
death. Although radiolabeled annexin V derivatives remain under
extensive investigation, their current in vivo usefulness is found
to be limited. Low target-to-background ratios, and long biological
half-life with high uptake in liver and kidney initiated the
Molecular imaging of cell death processes can be a very useful
tool to assist in diagnosis of disease, monitoring of disease course
and treatment evaluation, as well as in development and evalua-
tion of new drugs. Currently, most apoptotic cell death imaging ap-
proaches are based on targeting phosphatidylserine (PS). In most
⇑
Corresponding author. Tel.: +1 520 626 4248; fax: +1 520 626 2892.
E-mail address: zliu@radiology.arizona.edu (Z. Liu).
Present address: University Hospital Antwerp, Department of Nuclear Medicine,
Wilrijkstraat 10, B-2650 Oedeem (Antwerp), Belgium.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.029

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L. wyffels et al. / Bioorg. Med. Chem. 19 (2011) 3425–3433
development of conjugates with better biodistribution profiles.7
Although derivatives of annexin V continue to evolve, the so-called
second generation design is limited for protein-based probes like
annexin V.
robust complexes.25 The thermodynamic stability of the complexes
limits the presence of radiolabeled metabolites that may seriously
hamper in vivo imaging and quantification of the original tracer.26
HYNIC (2-hydrazinonicotinic acid), on the other hand, is one of the
most popular and efficient bifunctional chelators for 99mTc-labeling
of different groups of molecules.27 Therefore the HYNIC methodol-
ogy was chosen as an alternative strategy for 99mTc-labeling of
compound 1.
Low-molecular weight probes are more amenable to structural
optimization and display more favorable pharmacokinetic proper-
ties compared to protein-based probes like annexin V. Recently,
Smith et al. discovered that rationally designed bis(zinc(II)–
dipicolylamine) (Zn2+–DPA) coordination complexes can mimic
the apoptosis sensing function of annexin V.20 Two meta-oriented
Zn2+–DPA units are responsible for PS recognition and binding.12
Commercially available PSVueÒ794, with a Zn2+–DPA affinity
group conjugated to a near-infrared carbocyanine fluorophore
(ex. 794 nm, ex. 810 nm), was shown to selectively stain the same
cells as fluorescently labeled annexin V in cell culture.21 It was also
demonstrated that the probe selectively binds to anionic surfaces
of bacteria and can be used for in vivo imaging of bacterial infec-
tion in mice.22,23 Furthermore, PSVueÒ794 can be used for in vivo
imaging of necrotic regions in prostate and mammary tumor xeno-
grafts.21 Although NIR fluorophores are very useful for a wide array
of preclinical small animal imaging studies, clinical translation re-
mains at a proof-of-principle stage.24 The use of radioisotopes and
positron emission tomography (PET) and single photon emission
computed tomography (SPECT) imaging are more suited for deep
tissue imaging and clinical application. Since no radiolabeled
Zn2+–DPA coordination complex tracers have been described so
far, the aim of this study was radiolabeling of a Zn2+–DPA coordi-
nation complex (1) with SPECT isotope 99mTc and preliminary
in vivo and ex vivo evaluation of the usefulness of the new radio-
tracers for detection of apoptosis and necrosis.
In order to label the dipicolylamine subunits in compound 1
with 99mTc, the [99mTcꢀ(H2O)3(CO)3]+ precursor was first prepared
by addition of 99mTcO4 to an Isolink kit. Incubation of compound
1 (Fig. 1) with [99mTc(H2O)3(CO)3]+ at 100 °C for 20 min resulted in
[
99mTc(CO)3-1]3+ in P95% radiochemical yields. The concentration
of compound 1 needed to be 1 mM to achieve these high yields. Re-
verse phase HPLC (RP-HPLC) analysis of the reaction mixture is
presented in Figure 2. [99mTc(CO)3-1]3+ elutes from the column
with a retention time (tR) of 12.9 min. Following Sep-Pak purifica-
tion, radiochemical purities were consistently P98% by RP-HPLC
analysis.
HYNIC-1 was prepared by reacting compound 1 with the
N-hydroxysuccinimide (NHS) active ester of HYNIC in a 1:1 molar
ratio for 90 min at room temperature. Longer reaction times
(overnight) or higher concentrations of NHS-HYNIC did not signifi-
cantly increase the yield. Following purification, the identity of
HYNIC-1 was confirmed by mass spectrometry (MS) analysis.
99mTc-HYNIC-1 was prepared using conditions similar to those pre-
viously described.28 HYNIC-1 was reacted with 99mTcO4 in the
ꢀ
presence of stannous chloride and nicotinic acid/tricine as coligand
system at 90 °C for 20 min. HPLC analysis of the reaction mixture
revealed a high labeling efficiency with <2% of [99mTc]-pertechne-
tate (tR = 4.8 min) present (Fig. 2). However, besides the main peak
with a tR of 15.3 min (approx. 80%), some additional later-eluting
peaks (18.0 and 21.6 min, approx. 20% total) were observed that
may be related to isomerism or various numbers of coligands. The
presence of acetonitrile (MeCN) in the mobile phase used for HPLC
analysis can also result in mixed tricine/MeCN coligand complexes
on the column that are not present in the sample under analysis.29
HPLC purification of 99mTc-HYNIC-1 (isolation of the main peak at tR
of 14.9 min) did not significantly change in vivo behavior (data not
presented). Therefore, no attempts were yet made to optimize the
2. Results and discussion
2.1. Radiolabeling
Two radiolabeled compounds were prepared, [99mTc(CO)3-1]3+
and 99mTc-HYNIC-1 (Fig. 1). The [99mTc(CO)3]+ core was chosen
for labeling because of its simple aqueous chemistry. Tridentate
chelates like the dipicolylamine chelate moiety have been shown
to readily react with the [99mTc(CO)3]+ core to result in chemically
Figure 1. Structure of compound 1 and putative structures of [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1, complexed with Zn2+
.

L. wyffels et al. / Bioorg. Med. Chem. 19 (2011) 3425–3433
3427
Figure 2. Representative radiochromatograms of [99mTc(CO)3-1]3+ (A) and 99mTc-HYNIC-1 (B) before C18 Sep-Pak purification.
Table 1
2.3. Biodistribution study in normal mice
Stability of [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1 in aqueous solutions
A biodistribution study in normal BALB-c mice was done to
determine the influence of the 99mTc labeling strategy on tracer
tissue distribution. The distribution of radioactivity in various tis-
sues as a function of time following intravenous (iv) administration
of [99mTc(CO)3-1]3+and 99mTc-HYNIC-1 is represented in Figure 3.
Both tracers showed rapid blood clearance with [99mTc(CO)3-1]3+
demonstrating the fastest clearance (0.3 0.1% injected dose (ID)/
g versus 3.6 0.3%ID/g in blood at 1 h post injection (pi) for
Duration of incubation
0 h
2 h
4 h
20 h
[
99mTc(CO)3-1]3+ (%)
Saline
Serum
Cysteine
99mTc-HYNIC-1 (%)
Saline
Serum
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
98.2
99.9
77.7
82.3
80.7
75.9
81.5
80.5
74.8
80.7
80.4
72.1
66.7
56.3
[
99mTc(CO)3-1]3+and 99mTc-HYNIC-1, respectively). This rapid
Cysteine
blood clearance may substantially reduce background activity in
imaging studies, but might interfere with localization of the tracer
at the target site.
nd: not determined.
For both tracers, the highest uptake was in liver and intestines.
Liver uptake remained high throughout the study for
[
99mTc(CO)3-1]3+ (31.5 2.8%ID/g at 15 min and 19.3 1.8%ID/g at
radiolabeling procedure. The radiolabeling yield varied between
60% and 79%.
24 h pi). 99mTc-HYNIC-1 showed an initial high liver uptake
(22.0 2.8%ID/g at 15 min pi), followed by a continuous decrease
until the end of the study (4.5 0.8%ID/g at 24 h pi). The higher li-
ver retention of [99mTc(CO)3-1]3+ might be associated with the
somewhat higher lipophilicity of the compound compared to
99mTc-HYNIC-1. High liver uptake associated with the
99mTc(CO)3-core has been previously described by others.25,26,31
However, since the log P value of [99mTc(CO)3-1]3+ indicates only
low lipophilicity, log P value is most likely only one of the deter-
mining factors in the liver retention of [99mTc(CO)3-1]3+. Other fac-
tors like binding to plasma proteins, overall charge and complex
stericity may govern the non-specific liver retention.
2.2. In vitro stability and distribution coefficient
[
99mTc(CO)3-1]3+ and 99mTc-HYNIC-1 were analyzed by RP-HPLC
for stability after incubation in saline at room temperature and ser-
um at 37 °C for 20 h. The results are summarized in Table 1. The
tracers were analyzed for stability after addition of Zn(NO3)2. The
added Zn2+ did not seem to compete with the radioisotopes for
complexation. [99mTc(CO)3-1]3+ showed a high stability towards
incubation in saline and serum with P98% intact [99mTc(CO)3-1]3+
after 20 h of incubation. 99mTc-HYNIC-1 (main peak approx. 80%
at 0 h) was found to be less stable in saline and serum (72% and
67%, respectively, at 20 h). The main impurity that could be
detected was free 99mTc in the form of pertechnetate eluting at a
tR of 4.8 min. A challenge experiment with cysteine was also done.
The radiochemical purity of [99mTc(CO)3-1]3+ remained unchanged
when incubated in 100 mM cysteine solution for 20 h. The stability
of 99mTc-HYNIC-1 towards cysteine challenge was lower. The area
under the main peak diminished (56% at 20 h) while a peak at tR
4.8 min (6% at 20 h) and a peak at tR 8.4 min (17% at 20 h) increased.
The decrease in radiochemical purity can probably be related to
transchelation to cysteine.
Only minor uptake could be detected for both tracers in kid-
neys (3.6 0.4%ID/g at 1 h pi and 2.1 0.1%ID/g at 4 h pi for
[
99mTc(CO)3-1]3+ and 2.3 0.8%ID/g at 1 h pi and 1.2 0.2%ID/g at
4 h pi for 99mTc-HYNIC-1) and in urine (2.1 1.7%ID at 1 h pi and
0.03 0.03%ID at 4 h pi for [99mTc(CO)3-1]3+ and 2.4 3.3%ID at
1 h pi and 0.2 0.2%ID at 4 h pi for 99mTc-HYNIC-1) indicating
clearance of both tracers mainly through the hepatobilary system.
A higher uptake in lungs could be detected for 99mTc-HYNIC-1
compared to [99mTc(CO)3-1]3+ (2.3 0.6%D/g and 1.0 0.3%ID/g at
1 h pi for 99mTc-HYNIC-1 and [99mTc(CO)3-1]3+, respectively). Low
and comparable uptake could be detected in stomach and muscle
for both tracers.
The distribution coefficient of the tracers was determined in a
mixture of n-octanol and phosphate buffered saline (PBS, pH 7.4).
Log D values of ꢀ0.22 0.01 and ꢀ2.30 0.01 were measured for
Compared to 99mTc-labeled forms of annexin V, both tracers
showed lower kidney uptake and faster blood clearance.19 This fas-
ter blood clearance was to be expected considering the lower
molecular weight of our tracers compared to annexin V tracers. Li-
ver uptake, on the other hand, was higher for both DPA compounds
compared to 99mTc-labeled annexin V. Low-molecular weight
[
99mTc(CO)3-1]3+ and 99mTc-HYNIC-1, respectively. The lower
hydrophilicity of
[
99mTc(CO)3-1]3+ can be assigned to the
99mTc(CO)3-core which is known to increase the hydrophobicity of
complexes.30,31

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L. wyffels et al. / Bioorg. Med. Chem. 19 (2011) 3425–3433
Figure 3. Tissue distribution (%ID/g) at different time points post injection of [99mTc(CO)3-1]3+ (A) or 99mTc-HYNIC-1 (B) in normal BALB-c mice (n = 3 per time point).
probes like the Zn2+–DPA coordination complexes however, in con-
trast to large protein ligands like annexin V, are more amenable to
second generation design to lower the unfavorable liver uptake
and improve the imaging characteristics.32
did not show any staining of cleaved caspase-3, a biological indica-
tor of early apoptosis (Fig. 4). On the other hand, high levels of
stained cleaved caspase-3 were detected in the livers of mice trea-
ted with anti-Fas mAb, indicating the presence of massive hepatic
apoptosis. Tracer uptake in livers of control mice and anti-Fas trea-
2.4. Tracer uptake in mice with Fas-mediated hepatic apoptosis
ted mice was analyzed by planar scintigraphy, c-counting and by
autoradiography. Consistent with the results obtained in the bio-
distribution study in healthy mice, the highest uptake was de-
tected in livers of mice injected iv with [99mTc(CO)3-1]3+ (Fig. 5).
Uptake of [99mTc(CO)3-1]3+ was significantly (p <0.05) higher
(1.73 times) in livers of mice treated with anti-Fas mAb
(43.23 9.43%ID/g) compared to controls (26.85 3.88%ID/g). A
2.2 times higher uptake was detected in livers of anti-Fas mAb
treated mice (19.60 1.72%ID/g) injected with 99mTc-HYNIC-1
as opposed to livers of control mice (8.97 2.80%ID/g) (Figs. 4
and 5). The results are comparable to the increase in liver uptake
A well-described in vivo model of programmed cell death19,33,34
was used to determine the targeting properties of [99mTc(CO)3-1]3+
and 99mTc-HYNIC-1 for in vivo apoptotic cells. Fas is a cell-surface
receptor belonging to the death-receptor family.35 Intravenous
administration of an agonistic anti-Fas mAb will induce rapid
and massive hepatic apoptosis.33 To demonstrate presence of
apoptosis, routine H&E staining and immunohistological analysis
using a rabbit anti-cleaved caspase-3 was performed on 3
lm
slices of control and anti-Fas treated livers. Livers of control mice
Figure 4. Planar images 50 min after 99mTc-HYNIC-1 injection in anti-Fas treated (D) or normal mice (E). Liver, intestines and bladder are indicated by L, I and Bl. 60 min pi
livers were excised, and analyzed by autoradiography (A: anti-Fas treated, F: normal), H&E (B:anti-Fas treated, G: normal) and immunostaining (C: anti-Fas treated, H:
normal) using an anti-cleaved caspase-3 mAb (brown color in C).

L. wyffels et al. / Bioorg. Med. Chem. 19 (2011) 3425–3433
3429
and the phosphate anions of the PS headgroup. In
99mTc(CO)3-1]3+, on the other hand, one of the zinc ions is re-
[
placed by a 99mTc-tricarbonyl core. This saturated coordination
complex can only interact with the PS headgroups through weaker
electrostatic interactions, resulting in a lower affinity. Although
reasonably selective binding of anionic membranes has been re-
ported for compounds containing just a single Zn2+–DPA unit,37
the affinity would still be lower compared to 99mTc-HYNIC-1 con-
taining two Zn2+–DPA binding units.
The increase in liver uptake in anti-Fas treated mice might be
related to tissue damage caused by the anti-Fas mAb. Therefore,
inactivated control compounds with a minimally altered structure
and the molecular weight of [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1
are desirable to further investigate non-specific uptake.
Figure 5. Uptake of radioactivity (%ID/g) in livers of control mice or mice treated
with anti-Fas mAb, 60 min post iv injection of [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1.
2.5. Ex-vivo specificity for myocardial ischemia-reperfusion
injury
described for 99mTc-labeled annexin V derivatives19,36 and indicate
in vivo affinity of [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1 for hepatic
apoptosis. The best results are obtained for 99mTc-HYNIC-1 and can
be explained by a difference in binding strength. Like fluorescent
Zn2+–DPA coordination complexes,20,37 99mTc-HYNIC-1 presum-
ably binds to PS on the surface of apoptotic cells through coordina-
tion interactions between the two zinc ions and the carboxylate
A rat model of myocardial ischemia-reperfusion injury was
used as a second animal model to demonstrate sensitivity of the
99mTc-labeled tracers for apoptotic and necrotic tissue. Cell death
in this clinical situation occurs both in the ischemia as well as
the reperfusion phase.
H&E staining of the heart slices demonstrated cell damage in
the area at risk, characterized by contraction bands, wavy fibers,
Figure 6. Digital photographs of 2 mm myocardial slices from rats injected iv with 111 MBq of [99mTc(CO)3-1]3+ (A) or with 99mTc-HYNIC-1 (C). Normal myocardium is
colored with Evans blue (blue-grey zones); the area at risk is pink-red. Corresponding autoradiograms with [99mTc(CO)3-1]3+ (B) or 99mTc-HYNIC-1 (D) uptake matching the
area at risk.

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L. wyffels et al. / Bioorg. Med. Chem. 19 (2011) 3425–3433
congestion of red blood cells and in some areas polymorphonu-
clear cell infiltration, indicating early inflammation. Only few
apoptotic cells could be demonstrated by cleaved caspase-3 stain-
ing. This is in contrast to previously reported studies that could
demonstrate the presence of apoptotic cells in prolonged ische-
mia-reperfusion injury, using different detection methods.38–40
Selective accumulation of radioactivity in the area at risk undergo-
ing cell death could be observed for both tracers on autoradio-
grams of myocardial sections (Fig. 6). Area at risk to normal
tissue uptake ratios were 3.82 for [99mTc(CO)3-1]3+ and 5.45 for
99mTc-HYNIC-1. These values are comparable to those described
for 99mTc-labeled annexin V derivatives using similar rat models
of myocardial ischemia-reperfusion injury.39,41 Although the up-
take in the area at risk was significantly higher (p <0.05) than in
normal myocardium for both tracers, values are too low
(0.15 0.07%ID/g in area at risk and 0.04 0.02%ID/g in normal
myocardium for [99mTc(CO)3-1]3+, 0.12 0.03%ID/g in area at risk
and 0.02 0.00%ID/g in normal myocardium for 99mTc-HYNIC-1)
for in vivo SPECT imaging. Also, compared to the total myocardial
uptake (0.10 0.03%ID/g and 0.06 0.01%ID/g for [99mTc(CO)3-1]3+
and 99mTc-HYNIC-1, respectively), high liver uptake was present
(2.58 0.34%ID/g and 0.76 0.10%ID/g for [99mTc(CO)3-1]3+ and
99mTc-HYNIC-1, respectively). This liver uptake could seriously
hamper in vivo imaging due to a high background signal, limiting
the usefulness of the tracers for in vivo imaging of myocardial
ischemia-reperfusion injury.
technetate (99mTcO4ꢀ) eluted from a 99Mo-99mTc generator, was
purchased from GE Healthcare (Phoenix, AZ). IsoLink kits for the
preparation of [99mTc(H2O)3(CO)3]+ were kindly provided by Covi-
dien (St Louis, MO). Mass spectrometry was performed on a Ther-
mo-Finnigan (San Jose, CA) LCQ mass-spectrometer with
electrospray-ionization probe. 1H nuclear magnetic resonance
(NMR) spectrometry was performed on a Bruker DRX-600 spec-
trometer (Department of Chemistry, University of Arizona). Chem-
ical shifts were recorded in ppm (d) from an internal
tetramethylsilane standard in chloroform-d3. High performance li-
quid chromatography (HPLC) purification and analyses of the com-
pounds were performed using a Waters Breeze system (Waters
Corp., Milford, MA) equipped with a Waters 1525 binary pump,
Waters 2489 UV/VIS detector (k = 254 nm and 260 nm) in series
with a Bioscan B-FC-1000 radiodetector (Bioscan Inc., Washington,
DC). Radioactivity of samples was measured in a well counter.
High-activity samples were measured in an ionization chamber
dose calibrator (Capintec CRC, Ramsey, NJ). Autoradiograms were
obtained using a FujiFilm BAS5000 Bio-Imaging Analysis System
(Fujifilm Medical Systems USA, Inc., Stamford, CT) and were ana-
lyzed using Multi-Gauge 3.0 software. The animal experiments
were performed in accordance with the Principles of Laboratory
Animal Care from the National Institutes of Health (NIH Publica-
tion 85–23, revised 1985) and were approved by the Institutional
Animal Care and Use Committee (IACUC) at the University of
Arizona.
It must be noted that even though both tracers show selective
accumulation in the area at risk, this does not necessarily warrant
interaction with apoptotic or necrotic cells. Myocardial injury is
known to be associated with vascular hyperpermeability and in-
creased interstitional space.42,43 Therefore uptake in the area at
risk might also be associated with passive diffusion of the tracer
in parallel with specific uptake. Further research using a non-selec-
tive inactivated probe will be needed to characterize this non spe-
cific uptake.
4.2. Synthesis
4.2.1. N,N0-(5-(4-aminobutoxy)-1,3-phenylene)bis(methylene)
bis(1-(pyridine-2-yl)-N-(pyridine-2-ylmethyl)methanamine) (1)
Compound 1 was synthesized as previously described.44
4.2.2. Hydrazinonicotinamido-1 (HYNIC-1)
HYNIC conjugated 1 was synthesized by reacting succinimidyl
6-hydraziniumnicotinate hydrochloride (NHS-HYNIC) (Solulink,
Inc. San Diego, Ca) with 1 in a 1:1 ratio. 1 (2 mg, 3.4
solved in a mixture of MeCN (50 L), DMF (50 L) and 0.1 M HEPES
(400 L). To this solution NHS-HYNIC (3.4 mol, 0.975 mg dis-
solved in 100 L anhydrous DMF) was added dropwise. The reac-
tion was stirred for 90 min at room temperature, protected from
light. The reaction was stopped by addition of 1 mL H2O and the
reaction mixture was sent over a C18 Sep-Pak cartridge (Waters
Corp., Milford, MA) washed with 5 mL of H2O, and eluted with
1 mL of ethanol. The solution was evaporated to dryness and the
lmol) was dis-
3. Conclusion
l
l
l
l
The aim of this study was to synthesize radiolabeled Zn2+–DPA
coordination complexes and characterize their potential as tracers
for in vivo detection of cell death. Zn2+–DPA coordination complex
1 was successfully radiolabeled with 99mTc via 99mTc(CO)3+ or via
HYNIC to form [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1, respectively.
The hepatic apoptosis model suggests that both [99mTc(CO)3-1]3+
and 99mTc-HYNIC-1 target cells undergoing cell death. Selective
accumulation of [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1 was observed
in myocardial ischemia-reperfusion injury. However, the absolute
tracer uptake in the myocardium with ischemia-reperfusion injury
remained low and might be less detectable by in vivo cardiac imag-
ing. Further research is needed to enhance the tracer uptake in the
apoptotic/necrotic tissue and to reduce the liver accumulation. In
addition, further studies using other in vitro and in vivo models of
cell death are necessary to determine the binding affinity and exact
binding mechanism to apoptotic and necrotic cells. Labeling with
other radioisotopes such as 111In for SPECT imaging, and 68Ga, 18F
and 11C for PET imaging should be investigated to further explore
the potential of radiolabeled Zn2+–DPA coordination complexes
for in vivo detection of cell death.
l
residue dissolved in 100
vantage Pro300 (Thomson Instrument Co., Oceanside, CA), C18,
250 mm x 4.6 mm, particle size 5 m, eluted with a linear solvent
lL MeCN and purified by RP-HPLC (BioAd-
l
gradient of 5–50% B over 30 min (A = H2O with 0.1% TFA, B = MeCN
with 0.1% TFA). HYNIC-1 was isolated and characterized by 1H
NMR and mass spectrometry. Yield: 10.3 2.1%. 1H NMR (CDCl3):
d = 1.92 (4H, m); 3.57 (2H, m); 3.77 (4H, s); 3.86 (8H, s); 4.07
(2H, t); 6.70 (3H, br s); 7.08 (1H, d); 7.35 (8H, m); 7.82 (5H, m);
8.35 (1H, d); 8.75 (4H, d). MS (ESI) m/z (% rel int.): 723.4 (100.0
[MH]+); 745.5 (75.0 [M+Na]+); 362.2 (8.0 [M+2H]2+).
4.3. Radiolabeling
4.3.1. [99mTc(CO)3-1]3+
The radiolabeling was accomplished in two steps. First the
4. Experimental
[
99mTc(H2O)3(CO)3]+ precursor complex was prepared by adding
1 mL of 99mTcO4ꢀ to an Isolink kit and heating for 20 min in boiling
water. After cooling, the [99mTc(H2O)3(CO)3]+ solution (400
L) was
neutralized with 0.1 N HCl solution (60 L) and combined with a
1 mM (400 L) solution of compound 1 in MeCN/phosphate buf-
fered saline, pH 7.4 (PBS) (50:50). The vial was heated to 90 °C for
4.1. General procedures and materials
l
l
Chemicals were purchased from Sigma-Aldrich Chemical Com-
pany unless otherwise stated and used as they were received. Per-
l

L. wyffels et al. / Bioorg. Med. Chem. 19 (2011) 3425–3433
3431
20 min. Both the labeling precursor [99mTc(H2O)3(CO)3]+ and[
rections were made for the mass difference and density between
the two phases. The partition coefficient D was calculated as:
[radioactivity (cpm/mL) in n-octanol/radioactivity (cpm/mL) in
PBS]. The log D values are reported as an average of four different
measurements.
99mTc(CO)3-1]3+ were analyzed by RP-HPLC (BioAdvantagePro300
C18 5
l
m, 250 ꢁ 4.6 mm), using a 1 mL/min flow rate and the
following gradient: 0–3 min 100% H2O with 0.1% TFA, 3–5 min
100–90% H2O with 0.1% TFA, 5–12 min 90–0% H2O with 0.1% TFA,
12–25 min 100% MeCN with 0.1% TFA. To obtain an injectable solu-
tion, the radiolabeling mixture was passed through a C18 Sep-Pak
cartridge. The cartridge was washed with water (5 mL), and the
radiolabeled compound was eluted with ethanol (1 mL). The etha-
nol was reduced (<10%) by evaporation under a stream of nitrogen
and the radiolabeled compound was reconstituted in saline for
in vivo use.
4.7. Biodistribution study in normal mice
Biodistribution studies of [99mTc(CO)3-1]3+and 99mTc-HYNIC-1
were performed in normal BALB-c mice (n = 3 per time point) after
tail vein injection of 740 kBq of tracer. Mice were sacrificed by ip
injection of Beuthanasia-D (Schering-Plough Animal Health Corp.,
Union, NJ) under isoflurane anesthesia at 15 min, 1 h, 2 h, 4 h or
24 h post tracer injection. Blood was collected and main organs
to be examined were removed, rinsed in saline and weighed.
Radioactivity of the samples was measured in a well counter (Cap-
intec, Ramsey, NJ). Uptake is expressed as percentage of injected
dose (%ID) or percentage of injected dose/g of organ (%ID/g).
4.3.2. 99mTc-HYNIC-1
The following procedure was used to radiolabel hydrazinonic-
otinamide conjugated 1 with 99mTc. To approximately 50
l
g of
HYNIC-1 was added 0.5 mL of tricine solution (80 mg/mL in
PBS), 100 L of nicotinic acid solution (20 mg/mL in PBS), 20
l
lL
of SnCl2ꢂH2O (1 mg/mL in nitrogen purged 0.1 N HCl) and
0.3 mL of 99mTcO4
.
The mixture was heated for 20 min at
4.8. In vivo model of Fas-mediated hepatic apoptosis
ꢀ
90 °C. After cooling to room temperature, the reaction mixture
was purified by passing it through a C18-Sep-Pak cartridge. The
To demonstrate in vivo specificity of the tracers for apoptotic
cells, an in vivo mouse model of hepatic apoptosis was used.
BALB/c mice (n P 4 for each tracer) were injected iv under isoflu-
cartridge was washed with water (5 mL) to remove hydrophilic
impurities (99mTcO4
,
99mTc-co-ligand) and 99mTc-HYNIC-1 was
ꢀ
eluted with ethanol (1 mL). Ethanol was evaporated under a
stream of N2 and the radiolabeld compound was redissolved in
saline for in vivo evaluation. Radiochemical purity was assessed
rane anesthesia with purified hamster anti-Fas mAb (0.22 lg
anti-Fas mAb/g body weight, Jo2, BD Pharmingen, San Diego, CA).
90 min later, mice were injected iv with 850 kBq of [99mTc(CO)3-
1]3+ or 99mTc-HYNIC-1. In parallel, control mice (n P 4 for each
tracer) were injected with the same activity of radioligand. All ani-
mals were sacrificed 1 h pi using Beuthanasia-D (100 mg/kg) and
dissected. Organs were rinsed in saline to remove blood pool
activity and radioactivity was measured in a CRC-15W radioiso-
tope dose calibrator (Capintec, Ramsey, NJ) or in a well counter if
activity was <74 kBq. The uptake of radioactivity is expressed as
a percentage of the injected dose per gram of tissue plus or minus
the standard deviation (%ID/g tissue SD). Livers were quickly fro-
by analytical RP-HPLC (BioAdvantagePro300 C18
5 lm, 250
ꢁ 4.6 mm), using a linear solvent gradient of 5–50% B over
30 min (A = H2O with 0.1% TFA, B = MeCN with 0.1% TFA) and a
flow rate of 1 mL/min.
4.4. Preparation of Zn-complexes
In all experiments, purified 99mTc-HYNIC-1 was complexed with
Zn before use. A solution of 99mTc-HYNIC-1 in ethanol (1 equiv)
was mixed with Zn(NO3)2 (2 equiv) and stirred for 15 min at room
temperature. For in vivo evaluation, sterile saline was added to
reduce the ethanol concentration to <10%.
zen and cut into 100 lm thick slices (Cryostat Microm HM 550,
Thermo Scientific, Rockford, IL) and thaw-mounted on glass slides
for autoradiography.
4.5. Stability tests
4.9. In vivo imaging in Fas-mediated hepatic apoptosis
Reverse-phase HPLC analysis (BioAdvantage Pro300, C18,
Control and anti-Fas treated mice were imaged on a custom-
built planar imaging system (Radiology Department, University
of Arizona). The system uses a 25 mm-thick, low-energy parallel-
hole collimator with a bore spacing of 1.5-mm attached to a
114 mm-square modular gamma camera. The gamma camera uses
a 5 mm-thick NaI(Tl) monolithic scintillation crystal read out by a
3 ꢁ 3 array of photomultiplier tubes. Listmode data was collected
for 4 min and projections were formed using maximum-likelihood
position estimation techniques.45
250 ꢁ 4.6 mm, particle size 5
lm) was used to determine the sta-
bility of the radioligands towards incubation at 37 °C in rat serum
and towards incubation at room temperature in saline. Solid-phase
extraction (SPE) purified [99mTc(CO)3-1]3+ or 99mTc-HYNIC-1 was
diluted 20-fold in saline or rat serum and incubated at room tem-
perature or 37 °C, respectively. The stability of [99mTc(CO)3-1]3+
and 99mTc-HYNIC-1 was also tested in cysteine solution (100 mM
in PBS) at 37 °C. At t = 0 and after 2 h, 4, and 20 h incubation, sam-
ples of the solutions were analyzed by RP-HPLC. Gradient elution
as described above for [99mTc(CO)3-1]3+ and 99mTc-HYNIC-1 was
used.
4.10. Rat model of myocardial ischemia-reperfusion
A rat model of myocardial ischemia-reperfusion was used to ex-
plore uptake of the radioligands in ischemia-reperfusion injury.
Myocardial ischemia and reperfusion were produced in male Spra-
gue–Dawley rats (240–280 g) as previously described.46 The heart
was exposed by left thoracotomy. Myocardial ischemia was pro-
duced by ligation of the left coronary artery (LCA) for 30 min, fol-
lowed by 2 h reperfusion. At 2 h of reperfusion, rats were injected
iv with 111 MBq of [99mTc(CO)3-1]3+ (n = 4) or with 99mTc-HYNIC-
1 (n = 3). 2 h post tracer injection, rats were injected into the femo-
ral vein with Evans blue (2.5% in saline, w/v) to delineate the area at
risk (unstained) and euthanized using Beuthanasia-D (100 mg/kg).
Heart and main organs were rapidly removed, rinsed in saline to re-
4.6. Partition coefficient determination
To determine the partition coefficient (log Dn-octanol/PBS pH 7.4), an
aliquot of 20 l
L purified [99mTc(CO)3-1]3+ or 99mTc-HYNIC-1 was
added to a test tube containing 0.7 mL n-octanol (d = 0.827 g/mL)
and 0.7 mL PBS, pH 7.4. The tube was vortexed at room tempera-
ture for 2 min and then centrifuged for 10 min at 3000g (Biofuge
Pico, Heraeus Instruments, UK). Aliquots of 250 lL of the n-octanol
and the PBS phase were pipetted into tared test tubes, taking care
not to cross-contaminate between the two phases. The samples
were weighed and counted for radioactivity in a well counter. Cor-

3432
L. wyffels et al. / Bioorg. Med. Chem. 19 (2011) 3425–3433
move blood pool activity and blotted dry. Organs were weighed and
radioactivity was counted in a dose calibrator or well counter. The
heart was quickly frozen and sliced from apex to base, into 2-mm
thick transverse sections for autoradiography and histological anal-
ysis. The area at risk (AR), which was devoid of Evans Blue, was dis-
sected from the normal myocardium. After weighing the tissues,
tracer uptake (%ID/g) in the AR and the normal myocardium (N)
Analysis Shared Service (TACMASS), which is supported by the
Arizona Cancer Center Support Grant (NIH CA023074), for generat-
ing the immunohistochemical and histological data. This study was
supported by NIH grants NHLBI R01-HL090716 and NIBIB P41-
EB002035.
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