Synthesis, characterisation and evaluation of a novel copper-64 complex with selective uptake in EMT-6 cells under hypoxic conditions

Article abstract of DOI:10.1039/c3dt50960e

The radiometal ⁶⁴Cu is now widely used in the development of diagnostic imaging agents for positron emission tomography (PET). The present study has led to the development and evaluation of a novel chelating agent for ⁶⁴Cu: the new monothiourea tripodal ligand 1-benzoyl-3-{6-[(bis- pyridin-2-ylmethyl-amino)-methyl]-pyridin-2-yl}-thiourea (MTUBo). X-ray crystallographic analysis has shown this ligand forms a mononuclear complex with copper(ii) and co-ordinates via a trigonal bipyramidal N₄S array of donor atoms. Promisingly, cell uptake studies revealed that ⁶⁴Cu- MTUBo selectively accumulates in EMT-6 cells incubated under hypoxic conditions which may result from its relatively high Cu^II/I redox potential. Small-animal PET imaging and ex vivo biodistribution studies in EMT-6 tumor bearing BALB/c mice revealed significant tumor uptake after 1 h p.i., yielding tumor-to-muscle (T/M) and tumor-to-blood (T/B) ratios of 8.1 and 1.1, respectively. However, injection of ⁶⁴Cu-acetate resulted in similar uptake indicating that the observed uptake was most likely non-specific. Despite showing high in vitro stability, it is likely that in vivo the complex undergoes transchelation to proteins within the blood in a relatively short timeframe. For comparison, the hypoxia imaging agent ⁶⁴Cu-ATSM was also evaluated in the same murine tumor model and showed about 60% higher tumor uptake than ⁶⁴Cu-MTUBo. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3dt50960e

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Synthesis, characterisation and evaluation of a novel
copper-64 complex with selective uptake in EMT-6 cells
under hypoxic conditions†
Cite this: DOI: 10.1039/c3dt50960e
James C. Knight,a Melinda Wuest,a Fawaz A. Saad,b Monica Wang,a
David W. Chapman,a Hans-Soenke Jans,a Suzanne E. Lapi,c Benson M. Kariuki,d
Angelo J. Amoroso*d and Frank Wuest*a
The radiometal 64Cu is now widely used in the development of diagnostic imaging agents for positron
emission tomography (PET). The present study has led to the development and evaluation of a novel che-
lating agent for 64Cu: the new monothiourea tripodal ligand 1-benzoyl-3-{6-[(bis-pyridin-2-ylmethyl-
amino)-methyl]-pyridin-2-yl}-thiourea (MTUBo). X-ray crystallographic analysis has shown this ligand
forms a mononuclear complex with copper(II) and co-ordinates via a trigonal bipyramidal N4S array of
donor atoms. Promisingly, cell uptake studies revealed that 64Cu-MTUBo selectively accumulates in EMT-6
cells incubated under hypoxic conditions which may result from its relatively high CuII/I redox potential.
Small-animal PET imaging and ex vivo biodistribution studies in EMT-6 tumor bearing BALB/c mice
revealed significant tumor uptake after 1 h p.i., yielding tumor-to-muscle (T/M) and tumor-to-blood
(T/B) ratios of 8.1 and 1.1, respectively. However, injection of 64Cu-acetate resulted in similar uptake indi-
cating that the observed uptake was most likely non-specific. Despite showing high in vitro stability, it is
likely that in vivo the complex undergoes transchelation to proteins within the blood in a relatively short
timeframe. For comparison, the hypoxia imaging agent 64Cu-ATSM was also evaluated in the same
murine tumor model and showed about 60% higher tumor uptake than 64Cu-MTUBo.
Received 11th April 2013,
Accepted 28th June 2013
DOI: 10.1039/c3dt50960e
www.rsc.org/dalton
pharmacological processes with extremely high sensitivity
(10−11 to 10−12 mol L−1).
Introduction
Positron emission tomography (PET) is a non-invasive mole-
The success of PET imaging is dependent upon the use of
cular imaging technique which is now used routinely in pre- specifically designed molecular probes which have the ability
clinical and clinical settings to monitor the progression of to accumulate at particular targets associated with disease (e.g.
various diseases, including aggressive cancers.1 This technique up-regulated glucose metabolism,2 hypoxic microenvironment
offers important advantages compared with other commonly within tumors,3 and certain cellular or chemical biomarkers4).
used imaging modalities such as magnetic resonance imaging Consequently, the development of molecular probes contain-
(MRI) or computed tomography (CT). Most importantly, ing positron emitting radionuclides is an area of intense multi-
it has the ability to track biochemical, physiological and disciplinary research.1,5–7
At present, the most commonly used radionuclide for PET
imaging is fluorine-18 (t1/2 = 109.7 min; β+ = 0.64 MeV). Whilst
aDepartment of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada.
E-mail: wuest@ualberta.ca; Tel: +1 780 989 8150
this radionuclide has found tremendous utility in PET
imaging,8–11 the radioactive half-life of fluorine-18 is not com-
patible with high molecular weight targeting vectors (e.g.
monoclonal antibodies) which are well-known to have much
longer biological half-lives and extended blood clearance times
compared to small molecules. Consequently, much attention
has been focused on longer-lived metal radionuclides such as
bCollege of Applied Science, Chemistry Department, Umm Al-Qura University (UQU),
Makkah, Saudi Arabia. E-mail: fasaad@uqu.edu.sa
cDepartment of Radiology, Washington University School of Medicine,
510 S. Kingshighway Blvd, St. Louis, MO 63110, USA. E-mail: lapis@mir.wustl.edu;
Fax: +314 362 9940; Tel: +314 362 4696
dCardiff University of Wales – Department of Chemistry, Main Building, Park Place,
Cardiff, CF10 3AT, U K. E-mail: amorosoaj@cardiff.ac.uk; Fax: +44 (0)29 208 74030;
copper-64 (t1/2 = 12.7 h; β+ = 0.653 MeV, 17.4%; β−
=
Tel: +44 (0)29 208 74077
0.578 MeV, 39%) which has very favourable properties for
both diagnostic PET imaging and also radiotherapeutic
applications.12–14
†Electronic supplementary information (ESI) available. CCDC 931641. For
ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3dt50960e
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great promise is in the imaging and/or therapy of tumors
which contain regions of hypoxia.18–20 Hypoxic tissue (defined
as tissue with sub-normal concentrations of oxygen, pO2) is a
hallmark of certain cancers and is associated with poor prog-
nosis.21 In this case, the imaging agent 64Cu-ATSM3,22–25 is the
most successful in its class and is currently in phase II clinical
trials for imaging cervical cancer with PET.
Despite the clear value in the development of novel chelat-
ing agents for copper-64, the number and variety of com-
pounds that have been evaluated for this purpose has been
remarkably limited.13 These are mostly confined to either poly-
amino (tri- and tetraaza) macrocycles with pendant carboxylate
or thiolate arms for complexes with high in vivo stability and
resistance to transchelation, or acyclic tetradentate chelators
based on bis(thiosemicabazonato) ligands for imaging of
hypoxia. Some notable exceptions, including 64Cu-tachpyr
which features a tripodal array of pyridyl donors attached to a
rigid cyclohexyl backbone, have demonstrated the value in
exploring alternative structural motifs.26
Fig. 1 A selection of commonly used chelating agents for copper-64.
In this paper, a tripodal chelator derived from the well-
known ligand tris(2-pyridylmethyl)amine (TPMA; also often
referred to as TPA) was prepared and evaluated. TPMA is com-
monly used as a structural mimic of metal binding sites in
several endogenous proteins, including superoxide dismut-
ase.27 The ligand under investigation, referred to herein as
MTUBo, is functionalized at the 6-position of one of the
An important requirement for copper-64 is the use of an
appropriate chelating agent (Fig. 1). In many applications,
bifunctional chelators (BFCs) are used which have the ability
to (i) sequester the copper-64 cation within a thermodynami-
cally stable and kinetically inert framework, and (ii) undergo a
bioconjugation reaction (i.e. to form a covalent attachment)
with a selected targeting vector.12,15 Currently, the most
pendant pyridines with
a
benzoylthiourea attachment
(Scheme 1). Based on earlier investigations into the co-ordina-
tion chemistry of related ligand frameworks,28 it was antici-
pated that MTUBo would form a mononuclear 5-coordinate
trigonal bipyramidal copper(II) complex with an N4S donor set.
This is a very uncommon co-ordination environment com-
pared with other copper-64 complexes and consequently its
biological behaviour in relation to other standard compounds
such as 64Cu-ATSM was of interest.
popular chelating agent for this purpose is NOTA (log KCuL
=
21.6),16 however in vivo transchelation remains a major issue,
particularly in the liver where high levels of uptake are fre-
quently observed.17
Another area of investigation in which radioisotopes of
copper (including 60Cu, 61Cu, 62Cu and 64Cu) are showing
Scheme 1 Chemical synthesis of MTUBo.
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influences on complex stability have been reported to raise the
potential of the CuII/I redox couple, therefore making CuII
more easily reduced. Cu-MTUBo contains several structural
Results and discussion
Synthesis of ligand and copper(II) complex
The precursor compound mono(6-amino-2-pyridylmethyl)- features which are likely to contribute to a high CuII reduction
(2-pyridylmethyl)amine (MAPA) was synthesized as described potential. For example, tripodal ligands (including those
by Rivas et al.29 and Yamaguchi et al.30 MTUBo was prepared derived from TPMA) have been found to raise the reduction
by the reaction of MAPA with one equivalent of benzoyl iso- potential of CuII compared with macrocyclic analogues in
thiocyanate in ethanol to yield an oily crude material in some cases by a few hundred millivolts.43 The inclusion of
40 min which was difficult to transform into a solid despite sulphur donors within the inner co-ordination sphere of CuII
washing with several common solvents (chloroform, water, also tends to augment the reduction potential.40,43,44
dichloromethane, acetone, acetonitrile, diethyl ether,
The second redox couple observed in the cyclic voltammo-
dimethylsulphoxide (DMSO), or dimethylformamide (DMF)). gram of Cu-MTUBo is centered at −0.66 V vs. Fc+/Fc and is
However, washing with diethyl ether–acetone (1/1) led to a quasi-reversible. This is most likely attributable to a ligand-
white solid which was stable in air and not notably hygro- based process. In this case, the anodic and cathodic peak
scopic. The synthetic route to MTUBo is shown in Scheme 1.
potential difference (ΔE = 95 mV) is slightly larger than
The copper complex was readily formed via the dropwise expected for a fully reversible one-electron process, even when
addition of an acetonitrile copper(II) perchlorate solution to compared to the peak separation of the ferrocene internal
the ligand which was dissolved in hot acetonitrile. Upon standard.
addition, the solution changed to a clear green solution indi-
cating formation of the complex, however no precipitation of
the complex occurred. The copper complex was isolated from
the solution by slow diffusion of diethyl ether into the aceto-
nitrile solution which resulted in green crystals suitable for
Crystal structure of Cu-MTUBo
The copper compound crystallizes in the triclinic space group
ˉ
P1 and contains a single complex within the asu (Fig. 2; Tables
1–4). Shape mapping analysis45,46 of the co-ordination sphere
X-ray crystallography. The crystalline sample of Cu-MTUBo was
subsequently used for all spectroscopic measurements.
Electrochemical studies
In the potential range of 0 to −1 V, the cyclic voltammogram of
Cu-MTUBo contains two reduction processes (Fig. S1†). The
first reduction process occurs at −0.403 V (ΔE = 73 mV) refer-
enced against ferrocenium/ferrocene (Fc+/Fc). Based on our
experimental conditions, this value converts to approximately
−0.003 V vs. SCE, and 0.241 V vs. NHE.31,32 This process was
found to be reversible and occurs at a potential which is
typical of a one-electron reduction of copper(II) when co-ordi-
nated by a N4S donor set.28,33,34 Consequently, we have tenta-
tively ascribed this process to the CuII/I reduction. The
separation between anodic and cathodic waves for this
complex is similar to that observed for ferrocene indicating
uncompensated resistance in the cell. This value is signifi-
cantly higher than values reported for copper complexes of
well-known chelating agents such as NOTA, DOTA and
CB-TE2A which have CuII/I redox potentials of −0.70, −0.74,
Fig. 2 The crystal structure of Cu-MTUBo clearly shows the trigonal bipyrami-
and −0.88 V (vs. NHE), respectively.13
dal N4S coordination geometry around the metal centre. Displacement ellipsoids
are shown at 50% probability. H atoms are of arbitrary size.
Interestingly, the CuII/I redox potential of Cu-MTUBo is also
considerably higher than that of Cu-ATSM which appears at
−0.6 V vs. Ag/AgCl (−0.645 vs. SCE, and −0.401 V vs. NHE).35–37
Table 1 Selected bond lengths (Å) and angles (°) for [Cu-MTUBo]2+·2[ClO4−]-
For 64Cu-ATSM, the one electron reduction of the 64CuII cation
[CH3CN][H2O]
within a cellular environment is a key factor which leads to the
selective accumulation of radioactivity in regions of hypoxia. Cu(1)–N(1)
2.016(5)
2.007(5)
2.036(5)
Cu(1)–N(6)
Cu(1)–S(1)
2.088(5)
2.2615(17)
Cu(1)–N(2)
While the precise mechanism responsible for this selectivity is
Cu(1)–N(5)
still disputed, several recent studies have helped clarify this
process.38,39
N(1)–Cu(1)–N(5)
82.7(2)
81.8(2)
175.06(16)
82.5(2)
129.3(2)
N(2)–Cu(1)–N(6)
N(2)–Cu(1)–S(1)
N(5)–Cu(1)–N(6)
N(5)–Cu(1)–S(1)
N(6)–Cu(1)–S(1)
110.9(2)
94.21(14)
114.4(2)
96.66(16)
102.90(14)
It is known that the potential of the CuII/I redox couple is
N(1)–Cu(1)–N(6)
N(1)–Cu(1)–S(1)
N(2)–Cu(1)–N(1)
largely dependent upon the overall stability of the CuII
complex formed.40–42 Various structural features with negative N(2)–Cu(1)–N(5)
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complexation reactions were performed in DMSO. The most
effective method of preparation involved adding 15 µL of
64Cu(OAc)2 (typically in the range of 9–18 MBq) to 10 µg of
MTUBo in 85 µL of DMSO (prepared from a 1 mg mL−1 stock
solution). Incubation at 37 °C resulted in quantitative for-
mation of the copper-64 complex after 15 min. For animal
injections, saline (0.9%) was added to the DMSO solution
leading to a final solvent composition of 10% DMSO in saline.
The resulting solution was then passed through a syringe filter
to remove any insoluble particulates. In this case, the mean
decay-corrected radiochemical yield amounted to 75 6% with
a typical radiochemical purity exceeding 98%. It is worth
noting that when using higher amounts of MTUBo (1 mg) the
vast excess of unreacted ligand would precipitate instantly
upon the addition of saline. It was found that passing this
solution through a syringe filter yielded a clear solution
with no visible particles left which retained most of the
radioactivity.
Table 2 H-bonding geometry (Å) for Cu-MTUBo (with su’s except fixed and
riding H #1 x − 1, y, z, #2 x + 1, y, z).
D–H⋯A
D–H
H⋯A
D⋯A
D–H⋯A
N3–H3A⋯O1
0.88
0.88
0.92(1)
0.92(1)
0.92(1)
1.81
2.03
1.99(4)
2.14(2)
2.49(5)
2.559(6)
2.896(7)
2.848(14)
3.038(13)
3.177(16)
141.2
167.1
155(7)
167(6)
132(5)
N4–H4A⋯O10#1
O10–H1w⋯O5A
O10–H2w⋯O9A#2
O10–H2w⋯O8A#2
Table 3 Continuous symmetry mapping result of Cu-MTUBoa
Structure
1
(PP)
(VOC)
5.347
(TBPY)
1.473
(SPY)
3.926
(JSPY)
5.347
(JTBP)
3.437
33.736
a PP: pentagon (D5h), VOC: vacant octahedron (C4v), TBPY: trigonal
bipyramid (D3h), SPY: square pyramid (C4v), JSPY: Johnson square
pyramid (C4v), JTBP: Johnson trigonal bipyramid (D3h).
A log P value of −0.10 was determined for 64Cu-MTUBo
which indicates the dicationic complex is only slightly hydro-
philic. However, this value is notable as most dicationic
copper(II) complexes are very hydrophilic. A series of dicationic
monooxo-tetraazamacrocyclic copper-64 complexes reported
by Sun et al. were found to have log P values ranging from
−1.37 0.24 to −2.35 0.36.49 In contrast, 64Cu-ATSM with a
log P value of 1.48 is much more lipophilic.3
Table 4 Crystal structure data for Cu-MTUBo
Chemical formula
Colour/shape
CuC28H29Cl2N7O10
Green/plate
790.08
Triclinic
ˉ
P1
10.0397(9)
12.7362(9)
13.7012(10)
96.995(5)
90.820(4)
110.356(4)
1627.3(2)
2
S
Mr (g mol−1
)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
In vitro stability and cell uptake of 64Cu-MTUBo
Firstly, 64Cu-MTUBo was incubated in fetal bovine serum at
37 °C for 48 h. Radio-TLC analysis revealed the complex
remained almost entirely (>92%) intact with no observable
transchelation to serum proteins (data not shown).
γ (°)
V (Å3)
Z
Unique reflections
Observed reflections
Rint
7343
4679
0.0469
64Cu-MTUBo was also challenged independently with the
standard chelating agents NOTA, DOTA, CB-TE2A and
DiAmSar at 25 °C. In each challenge experiment, MTUBo and
the respective chelating agent were used in equimolar
amounts (5 mM). In the case of incubation with NOTA, DOTA
and CB-TE2A, no evidence of transchelation within 2 h was
observed. However, when incubated with DiAmSar, almost
complete transchelation was observed within 10 min. This
highlights the extraordinary thermodynamic stability and
rapid kinetic formation offered by cryptand species in the for-
mation of copper(II) complexes (Fig. S3†). From these experi-
ments, it can be concluded that 64Cu-MTUBo exhibits high
in vitro stability and a good ability to resist transchelation.
Finally, since the CuII/I redox potential of Cu-MTUBo was
found to be higher than Cu-ATSM (and therefore also likely to
be reduced within a cellular environment), 64Cu-MTUBo was
also evaluated in terms of its ability to accumulate in EMT-6
cells under hypoxic conditions. Encouragingly, we observed an
approximately 4.5-fold higher level of uptake in hypoxic versus
normoxic cells (Fig. 3). For comparison, we also evaluated
64Cu-ATSM which exhibited a similar (5-fold) cellular uptake
ratio between hypoxic versus normoxic cells, although on
R1[I > 2σ(I)]
wR2 (all data)
0.0881
0.2307
surrounding the CuII cation clearly indicates a trigonal bipyra-
midal geometry (S(TBPY) = 1.473; Table 3) which consists of
N1, N2, N5, N6 and S1. The four nitrogen bonds range
between 2.007(5) Å and 2.088(5) Å and the sulphur bond is sig-
nificantly longer at 2.2615(17) Å. The bond lengths and angles
within this structure are within the expected ranges on com-
parison to related structures, particularly those based on
TPMA such as the previously reported bisthiourea analogue
which also exhibited a trigonal bipyramidal N4S coordination
environment.28 A survey of the Cambridge Structural Database
(CSD)47 revealed 57 crystal structures of CuII with TPMA-based
ligands. All of these are five co-ordinate except one structure
for which the copper(II) cation has a distorted octahedral geo-
metry.48 In the case of Cu-MTUBo, an intramolecular hydrogen
bond is observed between the hydrogen of the amide and the
oxygen of the carbonyl (O1⋯H3A) at 1.82 Å (Table 2).
Radiosynthesis of 64Cu-MTUBo
Due to the poor solubility of the neutral MTUBo ligand in a a much higher overall uptake level. We postulate that as a
variety of aqueous buffers and ethanol, the copper-64 consequence of its higher lipophilicity, 64Cu-ATSM is able to
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Fig. 5 Radioactivity time activity curves in muscle (top) and tumor tissue
(bottom) after injection of 64Cu-MTUBo, 64Cu-ATSM, and 64Cu-actetate. Left:
first 20 min and right: 60 min plus the 24 h time point are shown.
Fig. 3 Uptake of 64Cu-ATSM and 64Cu-MTUBo into EMT-6 cells under normoxic
and hypoxic conditions. Radiotracers were incubated for 2 h. Data as mean
SEM.
with previous reports, 64Cu-ATSM was found to undergo both
hepatobiliary and renal clearance.22 The more hydrophilic
permeate through the cell membrane at a faster rate compared 64Cu-MTUBo appears to accumulate in the bladder to a higher
to 64Cu-MTUBo.
degree, indicating a more significant renal component to its
elimination. 64Cu-MTUBo also differs from 64Cu-ATSM in its
inability to cross the blood–brain barrier which is also most
likely due to its more hydrophilic character.
PET imaging of 64Cu-MTUBo and 64Cu-ATSM in BALB/c mice
bearing EMT-6 tumors
Fig. 4 shows representative images of 64Cu-MTUBo and 64Cu-
ATSM after 1 h and 24 h p.i., respectively. In the case of 64Cu-
MTUBo, an SUV of 0.50 0.08 (n = 4) was detected in the
tumor tissue after 1 h p.i. compared to 0.89 0.04 (n = 3) for
Ex vivo biodistribution of 64Cu-MTUBo and 64Cu-ATSM in
BALB/c mice bearing EMT-6 tumors
64Cu-ATSM. The observed muscle uptake was similar for both The organ biodistribution of 64Cu-MTUBo and 64Cu-ATSM in
radiotracers: SUV1h 0.24 0.05 (n = 4) and 0.16 0.03 (n = 3), EMT-6 tumor-bearing BALB/c mice is summarized in Table 5.
respectively. Time-activity curves for tumor and muscle uptake On comparison to well-known macrocyclic chelating agents
were determined for 64Cu-MTUBo and 64Cu-ATSM and directly such as DOTA,50 TETA,50 and CB-TE2A,51 the copper-64 com-
compared with 64Cu-acetate (Fig. 5). The results clearly illus- plexes of MTUBo and ATSM each reveal significantly higher
trate that injection of 64Cu-MTUBo and 64Cu-acetate into activity in the blood which does not appear to decrease over
EMT-6 tumor-bearing mice leads to a similar uptake level after time. This is most likely due to binding and/or transchelation
1 h p.i., while 64Cu-ATSM reaches a higher tumor uptake. to blood proteins. Analysis of the distribution of both 64Cu-
From these experiments, it can be concluded that after 1 h p.i. MTUBo and 64Cu-ATSM in the blood (between blood cells,
64Cu-MTUBo does not show any hypoxia-mediated uptake in plasma proteins, and supernatant plasma) at 1 h p.i. (Fig. 6)
this tumor model in vivo, while the difference between uptake supports this observation. Interestingly, a near-identical distri-
levels after injection of 64Cu-ATSM and 64Cu-acetate represents bution in the blood was found after injection of both radio-
the hypoxia-mediated fraction (Δ SUV ∼0.4). Interestingly, this labelled compounds with approximately 70% of the total
difference remains the same even after 24 h p.i. In accordance radioactivity in each case being bound to plasma proteins.
Fig. 4 Representative small animal PET images (maximum intensity projection) of EMT-6 tumor-bearing BALB/c mice after single intravenous administration of
∼5 MBq of 64Cu-MTUBo (left), 64Cu-ATSM (middle) or 64Cu-acetate (right).
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Metabolic profiling of 64Cu-MTUBo and 64Cu-ATSM
Table 5 Biodistribution (%ID/g
SEM) of 64Cu-MTUBo and 64Cu-ATSM in
BALB/c mice bearing an EMT-6 mammary tumor
It is notable that at 1 h p.i. 64Cu-MTUBo was eliminated intact
from the urine. However, when radio-TLC analysis of the
whole blood and the blood plasma was performed, the radio-
activity was found to remain on the baseline, indicating either
(i) complete metabolism of the radiolabelled compound, (ii)
complete transchelation of copper-64 to plasma proteins, or
(iii) complete binding of the intact complex to plasma pro-
teins. In addition, the tumors were harvested at 1 h p.i. and
the resulting homogenate was analysed by radio-TLC. Again, it
was found that all of the radioactivity remained on the
baseline.
64Cu-MTUBo
(1 h p.i.) n = 3
64Cu-MTUBo
(24 h p.i.) n = 4
64Cu-ATSM
(24 h p.i.) n = 4
T/M ratio
T/B ratio
Blood
Heart
Lung
8.30 2.32
1.13 0.28
2.15 0.21
8.39 2.36
2.65 0.17
23.00 0.87
22.41 3.89
15.28 6.58
3.74 0.68
3.06 0.81
9.41 2.70
9.21 0.33
4.20 0.61
2.53 0.39
0.42 0.20
0.30 0.02
2.43 0.60
1.31 0.09
0.20 0.03
0.47 0.17
14.42 2.79
2.60 0.20
1.97 0.03
8.25 0.56
3.07 0.38
16.27 0.57
9.33 1.55
1.36 0.18
3.94 0.51
2.43 0.29
4.14 0.50
4.05 0.32
4.90 0.44
1.64 0.30
0.90 0.08
0.39 0.06
5.10 0.33
0.72 0.02
0.40 0.01
0.23 0.05
18.15 0.76
3.12 0.26
1.33 0.08
2.66 0.31
4.18 0.38
12.54 1.31
7.44 0.79
1.57 0.37
2.95 0.62
1.83 0.12
2.10 0.30
4.35 0.21
4.30 0.14
1.32 0.09
0.79 0.09
0.23 0.03
4.17 0.49
0.89 0.17
2.61 0.33
0.16 0.03
Liver
Kidney (left)
Gallbladder
Spleen
Stomach
Duodenum
Small intestine
Large intestine
Pancreas
Bone
Muscle
Tumor
Ovaries
Brain
Taken together, identical behaviour for both 64Cu-MTUBo
and 64Cu-ATSM in blood plasma was observed at 1 h p.i.
Whilst some studies have found that 64Cu-ATSM does bind
intact to specific sites of human serum albumin,53 the present
data may indicate that due to the low stability of both analyzed
complexes they both also most likely undergo transchelation
to blood proteins. At this stage, no attempts were made to
further elucidate this behaviour.
Fat
Conclusions
Results from small animal PET imaging experiments involving
64Cu-MTUBo and 64Cu-ATSM showed slightly faster tumor
uptake in the case of 64Cu-ATSM. Uptake of radioactivity in
tumor tissue was similar for both 64Cu-MTUBo and 64Cu-
acetate indicating no specific enrichment under hypoxic con-
ditions. In the case of 64Cu-ATSM, while we clearly observe a
higher degree of tumor uptake, less than 50% of the overall
radioactivity in the tumor can be attributed to hypoxia-
mediated uptake with the remainder due to non-specific
uptake. For both 64Cu-MTUBo and 64Cu-ATSM, the radio-
activity in the blood was found to be largely protein-bound
after 1 h, and further analysis of the supernatant plasma (and
tumor homogenates in the case of 64Cu-MTUBo) revealed
Fig. 6 Distribution of 64Cu-MTUBo and 64Cu-ATSM in the blood at 1 h p.i.
For 64Cu-MTUBo, significant accumulation of radioactivity neither compound was in its original form. Whilst challenge
is observed at 1 h p.i. in the tumor, yielding tumor/muscle experiments performed in vitro suggest that 64Cu-MTUBo is
(T/M) and tumor/blood (T/B) ratios of 8.30 2.32 and 1.13
relatively resistant to transchelation, it can be hypothesized
0.28 (n = 3), respectively. At 24 h p.i., these values increase to that in an in vivo environment 64Cu-MTUBo either undergoes
14.42 2.79 and 2.60 0.20 (n = 4). No statistically different transchelation or binds intact to proteins in the blood in a
T/M and T/B ratios were found at 24 h p.i. for 64Cu-MTUBo relatively short timeframe. While significant tumor uptake was
and 64Cu-ATSM (Table 5).
clearly detectable in vivo following injection of 64Cu-MTUBo,
For both 64Cu-MTUBo and 64Cu-ATSM, a substantial level of direct comparison with 64Cu-ATSM and 64Cu-acetate suggests
radioactivity across all time points is located in the liver which this is mainly due to non-specific uptake.
is expected behaviour given the well-documented issue of
In the case of 64Cu-ATSM, there seems to be no apparent
transchelation to liver proteins such as superoxide dismu- correlation between lipophilicity and hypoxia selectivity.36,37
tase.52 This would also result from binding and/or transchela- However, the high lipophilicity of this complex is clearly a
tion to high molecular weight proteins in the blood. In both crucial property which allows it to quickly permeate through
cases, high levels of radioactivity are also located in the the cell membrane. 64Cu-MTUBo is comparatively more hydro-
kidneys at 1 and 24 h p.i., indicating a significant degree of philic in nature and it is therefore likely that this impedes the
retention. Unlike 64Cu-ATSM, 64Cu-MTUBo does not reveal sig- rate at which the complex crosses the cell membrane. While
nificant uptake in the brain.22 Overall, these observations 64Cu-MTUBo does not reveal any specific hypoxia-mediated
support the conclusions drawn from the small animal PET uptake in EMT-6 tumors in vivo, the promising results
experimental data.
obtained in vitro suggest some potential in this direction.
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procedures.58 Radio-TLCs were developed with methanol–10%
ammonium acetate 3/1 using aluminium-backed reverse phase
TLC plates (7.5 cm). These were analysed using a BAS-5000
Experimental
General
NMR spectra were measured on a Bruker AM-400 or Bruker reader and analysed using Aida software.
Av-500 or Bruker AC-250 Plus FT-NMR spectrometer. For infra-
red spectra, each compound was pressed into a disk with an Synthesis of MTUBo
excess of dried KBr and measured on a Jasco FT-IR spectrophoto-
MAPA (0.955 g, 3.131 mmol) was dissolved in ethanol (50 mL)
meter. Electrospray ionisation mass spectrometry (ESMS)
experiments were performed using a Waters LCT Premier XE
(oa-TOF) mass spectrometer. UV-VIS absorption spectra were
run in HPLC grade acetonitrile (Fisher) and measured on a
Jasco V-570 spectrophotometer from 230 to 1100 nm (optical
path length 1.0 cm). Elemental analyses were carried out by
the Warwick Analytical Service, University of Warwick.
and benzoyl isothiocyanate (0.421 mL, 3.131 mmol) was added
dropwise. The mixture was heated to 40 °C with stirring for
40 min then allowed to cool to room temperature. The solvent
was removed under reduced pressure to yield a brown oil
which was then treated with mixture of diethyl ether and
acetone to produce a white solid of MTUBo (0.447 g,
1
0.955 mmol) 30%. H NMR δH (250 MHz; CDCl3): 3.84 (2H, s);
Single crystal X-ray data were collected at 150 K on a Bruker/
Nonius Kappa CCD diffractometer using graphite monochro-
mated Mo-Kα radiation (λ = 0.71073 Å), equipped with an
Oxford Cryostream cooling apparatus. The data was corrected
for Lorentz and polarization effects and for absorption using
SORTAV.54 Structure solution was achieved by direct methods
(Sir-92 program system)55 and refined by full-matrix least-
squares on F2 (SHELXL-97)56 with all non-hydrogen atoms
assigned anisotropic displacement parameters. Hydrogen
atoms attached to carbon and nitrogen atoms were placed in
idealized positions and allowed to ride on the relevant carbon
atom and water hydrogens were refined with constrained geo-
metry. One perchlorate site (Cl2/Cl2a) is disordered (48/52%).
In the final cycles of refinement, a weighting scheme that gave
a relatively flat analysis of variance was introduced and refine-
ment continued until convergence was reached. Molecular
structures in the figures were drawn with ORTEP-3.0 for
Windows (version 2.02).57 Continuous Shape Mapping (CShM)
was used to calculate the deviation from the idealized poly-
hedron.45,46 CCDC 931641 contains the supplementary crystallo-
graphic data for this paper.
3.89 (4H, s); 7.14 (2H, t, Ar, J = 4.9 Hz); 7.42 (1H, d, Ar, J =
7.5 Hz); 7.50–7.76 (8H, m, Ar); 7.90 (2H, d, Ar, J = 7.5 Hz); 8.52
(2H, d, Ar, J = 4.1 Hz); 8.67 (1H, d, Ar, J = 8.1 Hz); 9.06 (1H, s,
NH); 13.05 (1H, s, NH). 13C δC (62.5 MHz; CDCl3): 59.3, 60.1,
114.0, 120.5, 121.9, 122.9, 127.4, 129.1, 131.6, 133.6, 136.4,
138.0, 149.0, 150.4, 158.6, 159.2, 166.3, 176.7. Accurate ESMS
(m/z): 491.1625 (30) [L1 + Na]+, 469.1805 (10) [L1 + H]+. [calcu-
lated 469.58]. C26H24N6O1S1. IR KBr/cm−1: ν = 3431 br, 1671 s,
1540 s, 1455 s, 1330 s, 1085 s, 624 s. UV/Vis [λmax, nm (ε M,
M−1 cm−1)] in CH3CN: 268 (62 300), 310 (33 500).
Synthesis of Cu-MTUBo
MTUBo (20 mg, 0.042 mmol) was dissolved in hot acetonitrile
(3 mL). To this stirring solution, the metal perchlorate salt
(1 equivalent) dissolved in acetonitrile (ca. 2 mL) was added
dropwise. Recrystallisation of the compound involved the
diffusion of diethyl ether into acetonitrile. The solution was fil-
tered through Celite® prior to recrystallisation.
WARNING: Perchlorate salts of metal complexes are poten-
tially explosive. Care should be taken while handling such
complexes.
The cyclic voltammetry experiments were carried out with a
PARSTAT 2273 potentiostat (Advanced Electrochemical System
from Princeton Applied Research) in conjunction with General
Purpose Electrochemical System software (GPES version 4.7 for
Windows) in a specially designed three electrode glass cell
[CuII(MTUBo)][ClO4]2·CH3CN·H2O. Green plate crystals
(45% yield). Found: C, 42.33; H, 3.51; N, 12.26%. CuC26H24-
N6O1S1(ClO4)2(CH3CN)(H2O) requires C, 42.57; H, 3.70; N,
12.41%; ESMS m/z (%): 530.0974 (100) [Cu(L1) − H]+, [calcu-
lated 530.0950]. IR (KBr pellet/cm−1): 3416(br), 1609(s),
1539(s), 1433(s), 1261(s), 1088(s), 625(s). UV/Vis [λmax, nm
(ε M, M−1 cm−1)] in CH3CN: 255 (13 100), 280 (8500),
326 (8700), 353 (5460), 631 (95), 822 (140), 950 (115).
with
a Teflon-coated cell cap. A Bioanalytical platinum
working electrode (model no. MF2013) with a 1.6 mm disk was
used for all experiments. The counter electrode was a platinum
wire and the reference electrode a non-aqueous Ag/AgNO3 elec-
trode. To regain electrochemical sensitivity and reproducibility
the working electrode was polished, with 600 grid emery
Radiosynthesis of 64Cu-MTUBo
paper, to a mirror surface and then ultrasonicated. Prior to To a solution of MTUBo (10 µg, 21 nmol) in DMSO (85 µL) was
each experiment, the electrode was washed with HPLC grade added 64Cu(OAc)2 (15 µL, typically 9–18 MBq) and the reaction
CH3CN and dried in air for about 15 min. A 0.1 M [Bu4N][PF6] mixture was placed in a thermoshaker (1000 rpm) at 37 °C for
solution in CH3CN was used as supporting electrolyte. In all 15 min. The progress of the reaction was monitored by reverse
cases, ferrocene was used as an internal reference. Solutions phase radio-TLC (Product Rf = 0.2). Upon completion of the
were degassed with nitrogen and a nitrogen atmosphere was complexation reaction (>98% radiochemical purity), saline
maintained over the solution during the experiment.
(0.9%, 900 µL) was added to the reaction mixture. The solution
Copper-64 was produced on a CS-15 biomedical cyclotron was passed through a 0.2 µm RC syringe filter (Phenomenex)
via the 64Ni(p,n)64Cu nuclear reaction at Washington Univer- and was then checked again by radio-TLC to ensure the radio-
sity School of Medicine (St. Louis, MO) according to published chemical purity exceeded 98%.
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Determination of the log P value for 64Cu-MTUBo
1 mL 5% trichloroacetic acid (TCA). The cell lysate was
counted in a Wizard gamma-counter (Wallace 1480 Wizard-3,
Perkin-Elmer, Woodbridge, Ontario, Canada). Protein levels
were quantified using a BCA protein assay kit (Pierce, Rock-
ford, IL, USA) according to the manufacturer’s recommen-
dations and bovine serum albumin was used as the protein
standard. Radiotracer cell uptake levels were normalized to
percent of the total added radioactivity per milligram protein
(% radioactivity per mg protein). Data are presented as means
SEM.
64Cu-MTUBo (5 µL) was added to a biphasic mixture consisting
of deionized water (1 mL) and octan-1-ol (1 mL). The mixture
was vortexed thoroughly for 5 min followed by centrifugation
at 1200 rpm for a further 5 min. A single portion of 200 µL was
taken from each layer using an auto pipette and transferred to
separate eppendorf tubes. The radioactivity in each eppendorf
tube was measured using a gamma scintillation counter. The
given value represents the mean average (n = 6).
Stability of 64Cu-MTUBo in fetal bovine serum (FBS)
Small animal PET imaging in BALB/c mice bearing EMT-6
tumors using 64Cu-MTUBo and 64Cu-ATSM
64Cu-MTUBo (ca. 300 kBq) in DMSO (100 µL) was added to FBS
(500 µL) and vortexed. The solution was then placed in a
thermoshaker (300 rpm) at 37 °C. At 10 min, 1 h, 24 h and
48 h, the solution was analysed by reverse phase radio-TLC to
determine the percentage of radioactivity bound to proteins.
All animal experiments were carried out in accordance with
guidelines of the Canadian Council on Animal Care (CCAC)
and were approved by the local animal care committee of the
Cross Cancer Institute. Positron emission tomography (PET)
experiments were performed using BALB/c mice bearing
EMT-6 tumors. The mice were not fasted prior to imaging
experiments. The animals were anesthetized through inhala-
tion of isoflurane in 40% oxygen/60% nitrogen (gas flow, 1 L
min−1) and body temperature was kept constant at 37 °C for
the entire experiment. Mice were positioned and immobilized
in the prone position with their medial axis parallel to the
axial axis of the scanner and their thorax, abdomen and hind
legs (organs of interest: heart, kidneys, bladder, liver) in the
centre of the field of view of the microPET® R4 scanner
(Siemens Preclinical Solutions, Knoxville, TN, USA). A trans-
mission scan for attenuation correction was not acquired. 4–6
MBq of either 64Cu-MTUBo or 64Cu-ATSM in 100–150 μL saline
(0.9%) containing 10% of DMSO (in the case of 64Cu-MTUBo)
or 10% of ethanol (in the case of 64Cu-ATSM) was injected
through a needle catheter into the tail vein. Data acquisition
Challenge experiments involving macrocyclic chelators
To a 5 mM solution of MTUBo in DMSO (145 µL) was added
64Cu(OAc)2 (5 µL, ca. 1 MBq). The eppendorf containing the
solution was placed into a thermoshaker (350 rpm) at 37 °C
for 15 min. An equimolar quantity of either: (i) DOTA, (ii)
NOTA, (iii) CB-TE2A, or (iv) DiAmSar in DMSO (150 µL) was
then added to the solution. The resulting solutions were vor-
texed thoroughly and then placed in a thermoshaker (350 rpm)
at 25 °C. At 10, 60 and 120 min, the reaction mixtures were
analysed by reverse phase radio-TLC to determine the extent of
each 64Cu complex in solution. These experiments were per-
formed in triplicate.
In vitro EMT-6 cell uptake experiments under hypoxic
conditions
For these experiments the mouse mammary tumor cell line continued for 60 min in 3D list mode. The frames were re-
EMT-6 (a gift from Dr David Murray, Dept. of Oncology, Univer- constructed using MAP. The pixel size was 0.085 by 0.085 by
sity of Alberta) was used. The cells were cultured at 37 °C and 0.12 cm and the resolution in the centre field of view was
5% CO2 (normal conditions) in Gibco DMEM-F-12 medium 1.8 mm. No correction for partial volume effects was per-
supplemented with 15 mM HEPES, L-glutamine and 1% peni- formed. The image files were further processed using the
cillin/streptomycin. For the radiotracer uptake studies cells ROVER v 2.0.21 software (ABX GmbH, Radeberg, Germany).
were grown in glass dishes (55 mm diameter) plated at a Masks for defining 3D regions of interest (ROI) were set and
density of around 500 000 cells per plate and incubated for the ROIs were defined by thresholding. ROI time-activity
24 h at normal conditions. Following that, half of the plates curves (TACs) were generated for subsequent data analysis.
were taken for culture under hypoxic conditions. Hypoxic cell Standardized uptake values (SUV = (activity per mL tissue)/
culture conditions were achieved using a special sealed (injected activity/body weight), mL g−1) were calculated for
chamber device (Div. of Experimental Oncology, Dept. of each ROI. Data are expressed as means SEM from n investi-
Oncology, University of Alberta), in which O2 can be nearly gated animals. All TACs were constructed using GraphPad
completely replaced with N2. The cells were cultured for Prism 4.0 (GraphPad Software, San Diego, CA, USA).
another 24 h under hypoxic conditions. For the radiotracer
Ex vivo biodistribution of 64Cu-MTUBo and 64Cu-ATSM in
incubation the medium was replaced with Krebs Ringer buffer
BALB/c mice bearing EMT6 tumors
(115 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4,
1.2 mM Na2SO4, 2.5 mM CaCl2, 25 mM NaHCO3 and 5 mM Biodistribution experiments were performed after the PET
glucose at a pH of 7.4) at room temperature. Around 0.2 MBq scans were finished. Animals were euthanized by cervical dis-
64Cu-radiotracer was added per plate and incubated for 2 h location and organs of interest harvested. Radioactivity in all
under either normal or hypoxic conditions. After incubation, tissue samples was measured in the gamma-counter. Data was
cells were rinsed twice with ice-cold Krebs-Ringer buffer to analyzed as percentage of the injected dose per gram tissue
stop further cell uptake and then immediately lysed using (% ID g−1) and is presented as means SEM.
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Distribution of 64Cu-MTUBo and 64Cu-ATSM in the blood
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Typically, 5 MBq of 64Cu-MTUBo or 64Cu-ATSM in 100–150 μL
of saline (0.9%) containing 10% of DMSO (in the case of 64Cu-
MTUBo) or 10% of ethanol (in the case of 64Cu-ATSM) was
injected as a bolus through a catheter into the tail vein of iso-
flurane anesthetized BALB/c mice bearing EMT-6 tumors.
Before radiotracer injection, mice were heparinized by sub-
cutaneous injection of 50 μL heparin (1000 I. U.) and kept under
anesthesia during the course of the experiment. At 60 min p.i.,
the animal was sacrificed and a whole blood sample (approxi-
mately 500 μL) and urine were collected. Blood cells were sep-
arated by immediate centrifugation (5 min at 13 000 rpm).
Proteins within the sample were precipitated by adding metha-
nol (ca. 800 μL) to the supernatant following a second centrifu-
gation step (5 min at 13 000 rpm). The activity present in each
fraction was measured using the gamma-counter.
Metabolic profiling of 64Cu-MTUBo and 64Cu-ATSM
For metabolic profiling, the supernatant plasma fractions and
the urine collected at 1 h p.i were analysed by reverse phase
radio-TLC as described above. In the case of 64Cu-MTUBo,
EMT-6 tumors were also harvested at 1 h and 24 p.i. for meta-
bolic profiling. The tumors were cut into small pieces and
homogenized in PBS (pH 7.4, 5 mL) for 5 min. The tumor
homogenates were then analysed by reverse phase radio-TLC.
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Acknowledgements
19 F. Dehdashti, P. W. Grigsby, M. A. Mintun, J. S. Lewis,
B. A. Siegel and M. J. Welch, Int. J. Radiat. Oncol. Biol.
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F. W. thanks the Dianne and Irving Kipnes Foundation,
Alberta Innovates – Health Solutions (AIHS), and the Natural
Sciences and Engineering Research Council of Canada
(NSERC) for supporting this work. We like to thank Tom Voller
from the Dept. of Radiology at Washington University School
of Medicine in St. Louis for the production and shipping
handling of Cu-64. In addition we like to thank Gail Hipperson
and Dan McGinn from the Vivarium at the Cross Cancer Insti-
tute for the animal handling.
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