Synthesis and characterization of a hypoxia-sensitive MRI probe

Article abstract of DOI:10.1002/chem.201200266

Tissue hypoxia occurs in pathologic conditions, such as cancer, ischemic heart disease and stroke when oxygen demand is greater than oxygen supply. An imaging method that can differentiate hypoxic versus normoxic tissue could have an immediate impact on therapy choices. In this work, the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a 2-nitroimidazole attached to one carboxyl group via an amide linkage was prepared, characterized and tested as a hypoxia-sensitive MRI agent. A control complex, Gd(DO3A-monobutylamide), was also prepared in order to test whether the nitroimidazole side-chain alters either the water proton T₁ relaxivity or the thermodynamic stability of the complex. The stabilities of these complexes were lower than that of Gd(DOTA)^- as expected for mono-amide derivatives. The water proton T₁ relaxivity (r ₁), bound water residence lifetime (τ_M) and rotational correlation time (τ_R) of both complexes was determined by relaxivity measurements, variable temperature ¹⁷O NMR spectroscopy and proton nuclear magnetic relaxation dispersion (NMRD) studies. The resulting parameters (r₁=6.38 mM^-1 s^-1 at 20 MHz, τ_M=0.71 μs, τ_R=141 ps) determined for the nitroimidazole derivative closely parallel to those of other Gd(DO3A-monoamide) complexes of similar molecular size. In vitro MR imaging experiments with 9L rat glioma cells maintained under nitrogen (hypoxic) versus oxygen (normoxic) gas showed that both agents enter cells but only the nitroimidazole derivative was trapped in cells maintained under N₂ as evidenced by an approximately twofold decrease in T₁ measured for hypoxic cells versus normoxic cells exposed to this agent. These results suggest that the nitroimidazole derivative might serve as a molecular reporter for discriminating hypoxic versus normoxic tissues by MRI. Hunting hypoxic cells: The DO3A-monoamide ligand with a 2-nitroimidazole moiety was synthesized with an aim to complex the Gd ^III ion as well as to target and visualize hypoxic cells by using the MRI technique (see figure). In vitro MRI experiments revealed that the conjugate might be suitable for assessment of hypoxia in vivo as the agent was selectively trapped in hypoxic (9L rat glioma) cells. Copyright

Full text of DOI:10.1002/chem.201200266

FULL PAPER
DOI: 10.1002/chem.201200266
Synthesis and Characterization of a Hypoxia-Sensitive MRI Probe
[
a]
[b]
[b]
Federico A. Rojas-Quijano, Gyula Tircsꢀ,* Enik o˝ Tircsꢀnꢁ Benyꢀ,
[
b]
[b]
[b]
[c]
Zsolt Baranyai, Huan Tran Hoang, Ferenc K. Kꢂlmꢂn, Praveen K. Gulaka,
[a, e]
[
c]
[d]
[a]
Vikram D. Kodibagkar, Silvio Aime, Zoltꢂn Kovꢂcs, and A. Dean Sherry*
Abstract: Tissue hypoxia occurs in
pathologic conditions, such as cancer,
ischemic heart disease and stroke when
oxygen demand is greater than oxygen
supply. An imaging method that can
differentiate hypoxic versus normoxic
tissue could have an immediate impact
on therapy choices. In this work, the
ivity or the thermodynamic stability of
the complex. The stabilities of these
tR =141 ps) determined for the nitro-
imidazole derivative closely parallel to
AHCTUNGTRENNUNG
complexes were lower than that of Gd-
those of other Gd(DO3A-monoamide)
complexes of similar molecular size. In
vitro MR imaging experiments with 9L
rat glioma cells maintained under ni-
trogen (hypoxic) versus oxygen (nor-
moxic) gas showed that both agents
À
A
H
U
G
R
N
U
G
derivatives. The water proton T relax-
1
ivity (r ), bound water residence life-
1
time (t ) and rotational correlation
M
time (t ) of both complexes was deter-
R
gadolinium
tetraazacyclododecane-1,4,7,10-tetra-
acetic acid (DOTA) with a 2-nitroimi-
dazole attached to one carboxyl group
via an amide linkage was prepared,
characterized and tested as a hypoxia-
sensitive MRI agent. A control com-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(III) complex of 1,4,7,10-
mined by relaxivity measurements, var-
enter cells but only the nitro AHCTUNGTERUNNNiG midazole
derivative was trapped in cells main-
1
7
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
iable temperature O NMR spectros-
copy and proton nuclear magnetic re-
laxation dispersion (NMRD) studies.
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
tained under N as evidenced by an ap-
2
proximately twofold decrease in T1
measured for hypoxic cells versus nor-
moxic cells exposed to this agent.
These results suggest that the nitroimi-
dazole derivative might serve as a mo-
lecular reporter for discriminating hy-
poxic versus normoxic tissues by MRI.
The
6.38 mm
resulting
parameters
(r =
1
À1 À1
s
at 20 MHz, t =0.71 ms,
M
plex,
Gd(DO3A-monobutylamide),
Keywords: imaging agents · imag-
ing of hypoxic tissue · ligand design ·
relaxation properties · stability
was also prepared in order to test
whether the nitroimidazole side-chain
alters either the water proton T relax-
1
Introduction
[
a] Dr. F. A. Rojas-Quijano, Prof. Z. Kovꢀcs, Prof. A. D. Sherry
Advanced Imaging Research Center
University of Texas Southwestern Medical Center
Tissue hypoxia usually reflects a locally restricted oxygen
supply (ischemia) and/or an abnormal increase in oxygen
consumption. In clinical practice, hypoxia is related to a vari-
ety of pathological conditions including cancer, ischemic
heart disease and stroke. Locally advanced solid tumors
often have hypoxic or anoxic regions distributed heteroge-
neously throughout the tumor as a result of a structurally or
functionally abnormal vasculature. Tumor hypoxia is com-
monly associated with a poor prognosis after radiation and
chemotherapy, and hypoxia can influence many aspects of
tumor proliferation, such as growth, apoptosis, angiogenesis
5
323 Harry Hines Boulevard, Dallas, Texas 75390 (USA)
Fax : (+1)214-645-2744
E-mail: dean.sherry@utsouthwestern.edu
[
b] Dr. G. Tircsꢁ, E. Tircsꢁnꢂ Benyꢁ, Dr. Z. Baranyai, H. Tran Hoang,
Dr. F. K. Kꢀlmꢀn
Department of Inorganic and Analytical Chemistry
University of Debrecen, Egyetem tꢂr 1
Debrecen, H-4010 (Hungary)
Fax : (+36)52-518-660
E-mail: gyula.tircso@science.unideb.hu
[
c] Dr. P. K. Gulaka, Prof. V. D. Kodibagkar
Department of Radiology
University of Texas Southwestern Medical Center
[1,2]
and metastasis.
In 1955, 2-nitroimidazole was shown to be active against
5
323 Harry Hines Boulevard, Dallas, Texas 75390 (USA)
[3]
certain anaerobic bacterial infections. Later it was discov-
ered that 2-nitroimidazole analogues could be used to sensi-
[
d] Prof. S. Aime
Department of Chemistry IFM & Molecular Imaging Center
Universitꢃ degli Studi di Torino
[4,5]
tize hypoxic tumors to ionizing radiation.
Further re-
Via P. Giuria 7, 10125 Torino (Italy)
search on the mechanism of action of this compound re-
vealed that it was capable of accumulating in hypoxic tissue
due to selective enzyme-mediated reduction of the nitro
[
e] Prof. A. D. Sherry
Chemistry Department, University of Texas at Dallas
5
323 Harry Hines Boulevard, Dallas, Texas 75390 (USA)
[6]
group under low oxygen conditions. The diverse bioactivity
of nitroimidazoles and related nitroaromatic compounds has
the same underlying mechanism; the activities of these com-
Fax : (+1)972-883-2925
E-mail: sherry@utdallas.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200266.
[7]
pounds depend on the redox potential of the nitro group.
Chem. Eur. J. 2012, 00, 0 – 0
ꢄ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
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The first step of this intracellular process involves the one-
electron reduction of the nitro group to a radical anion,
which undergoes further reduction in hypoxic cells but is
rapidly oxidized back to the nitro compound under normox-
ic conditions. The reduction potential of the nitro group in
nitroimidazoles is sensitive to its position in the imidazole
[8]
ring and this correlates with hypoxia sensitivity. Given the
prospect of nitroimidazole as a platform for hypoxia-homing
probes, several radiolabeled nitroimidazole derivatives and
related compounds have been tested as imaging probes of
[9]
hypoxic tissue, in vivo. Most prominent among theses have
1
8
18
[10]
been the F labeled fluoromisonidazole ( F-FMISO) for
9
9m
PET imaging and the
Tc labeled propylene amineoxime
BMS181321) for SPECT imaging. Cyclen (1,4,7,10-tetra-
ACHTUNGTRENNUNGa zacyclododecane)-based nitroimidazole derivatives tagged
[11]
(
67
III 153
III
177
III
with Ga , Gd or Lu have shown only moderate
Scheme 1. Synthetic route to ligand 3 (overall yield 28%).
[12–14]
tumor localization.
Although several multifluorinated
derivatives of 2-nitroimidazole have been synthesized and
1
9
used to assess tumor hypoxia by F NMR spectroscopy,
The protected DO3A-monoamide derivative 1 was ob-
tained by alkylating DO3A-tris(tert-butyl ester) with methyl
chloroacetate and treating the resulting methyl ester with
excess diaminobutane (putrescine) following a slight modifi-
cation of a published procedure (see the Supporting Infor-
III
1
Gd -based nitroimidazole derivatives for H MRI have not
[15–17]
been reported.
Although MRI is considerably less sensi-
tive than nuclear medicine for molecular imaging, it does
offer the advantage of wide clinical availability, lack of radi-
ation and high resolution anatomical images. For these reas-
[22]
mation). Nitroimidazoyl hexanoic acid (2) was synthesized
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
ons, we initiated a program to develop gadolinium-based 2-
by alkylation of 2-nitroimidazole with ethyl bromohexan-
nitroimidazole derivatives and to evaluate the potential of
such agents for detecting hypoxic cells by MRI.
ACHTUNGTRENNUNoG ate followed by the cleavage of the ethyl group in concen-
[23]
trated HCl, as previously reported. The imidazoyl acid 2
and macrocyclic amine 1 were then coupled by using stan-
[24,25]
dard HBTU/HOBt chemistry.
The tert-butyl protecting
Results and Discussion
groups of the intermediate ester were cleaved by using tri-
fluoroacetic acid (TFA) and the crude product was purified
by HPLC to give pure 3 (28% yield). Gd(3) was obtained in
Ligand design and synthesis: DOTA (1,4,7,10-tetraazacyclo-
dodecane-1,4,7,10-tetraacetic acid) was selected as the chela-
tor for the conjugation to 2-nitroimidazole because Gd
quantitative yield by treatment of 3 with GdCl at pH 6.
3
III
complexes with DOTA-like ligands have high thermody-
namic stability, are kinetically inert and have favorable re-
Ligand protonation and complex stability constants: The co-
ordination environment (four macrocyclic N-donor atoms,
three carboxylate O-donor atoms and one amide O-donor
atom) provided by ligand 3 is typical of a DOTA- and
DO3A-bioconjugate in which the chelator is attached to the
biomolecule through one of the acetate pendant arms of the
[18,19]
laxation properties.
The 2-nitroimidazole vector was se-
lected based on its one-electron reduction potential, known
to fall in the range of À330 to À450 mV, which allows for its
entrapment and exclusive reduction under hypoxic condi-
tions as it has been previously reported in the literature by
[20]
ligand.
Despite the widespread use of these ligands to
[7]
3+
many scientists. Since conjugation of DOTA to a targeting
vector via a single carboxyl group is relatively straightfor-
chelate Gd for MRI and various radioactive lanthanide
ions for radiopharmaceutical applications, only a limited
number of studies have appeared on the thermodynamic sta-
[20]
ward,
a 2-nitroimidazole moiety with an extended side-
chain containing a primary amino group was chosen for con-
jugation (Scheme 1). This design affords a neutral Gd
bility, formation and dissociation kinetics of DO3A-mono-
III
[26,27]
A
H
U
G
R
N
N
amides.
Here, we report the thermodynamic stabilities
1
complex, which would hopefully help facilitate cellular
uptake. It was anticipated that the relatively long linkage
between Gd(DO3A-monoamide) unit and the nitroimida-
zole moiety in 3 would minimize any unwanted interference
of the Ce(3), Gd(3) and Yb(3) and the results of H and
1
7
O NMR relaxometric studies of Gd(3). The results are also
compared to corresponding data for Ce(6), Gd(6) and
Yb(6).
III
between the Gd coordination sphere and the nitroimida-
Generally, pH potentiometry is considered the gold stan-
dard technique for determining the protonation constants of
polyaminopolycarboxylate ligands and thermodynamic sta-
bility constants of complexes formed with various metal
ions. The protonation constants obtained from fitting the
pH-potentiometric data for ligands 3 and 6 are presented in
Table 1 along with the literature data for DOTA (4) and
zole unit and, at the same time, increase complex lipophilici-
ty. In addition, the linker does not contain groups that can
engage in prototropic equilibria at physiological pH and
should therefore not affect the redox potential of the nitro-
[8,21]
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
imidazole unit.
The synthesis of the target ligand is out-
lined in Scheme 1.
&
2
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ꢄ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

A Hypoxia Sensitive MRI Probe
FULL PAPER
[
a]
Table 1. Protonation constants of 3 and some ligands of similar structure, 1m KCl, an increase in the length of the alkyl substituent
2
58C.
had a noticeable effect on the first protonation con-
stant (Table S1 in the Supporting Information).
The formation equilibria between these macrocy-
[
b]
[e]
[b]
[h]
logK
3
4
5
6
7
H
H
H
H
H
[c]
[d]
[f]
[g]
logK
logK
logK
logK
logK
1
2
3
4
5
9.78(2)
9.05(3)
4.53(4)
3.17(5)
2.19(6)
12.09; 12.6
9.67; 9.70
4.55; 4.50
4.09; 4.14
2.32
30.40; 30.94
9.6
9.2
4.4
1.7
–
10.17(1), 12.22, 10.73
9.02(1), 8.90, 9.05
4.41(1), 4.34, 4.53
2.94(1), 2.49, 3.32
1.99(1), 1.47, 2.25
26.54, 27.94, 27.63
9.56
5.95
–
–
–
[c]
[d]
[f]
[g]
[g]
[g]
[g]
III
clic ligands and the Ln ions cannot be studied by
[c]
[d]
[f]
direct pH potentiometric titration because of slow
formation kinetics. Hence, the stability constants
were determined by the use of the “out-of-cell”
technique with separate samples for each titration
[f] in point (total of 16 points). The stability constants of
[c]
[d]
[f]
[d]
[f]
[c]
[d]
[f]
[g]
[i]
logb
4
26.53
24.9
15.51
[
a] Standard deviations shown in parentheses; [b] this work, in KCl (1.0m) and at
[
32]
[33]
[26]
2
58C; [c] in KCl (0.1m);
[d] in Me
4
NCl (1.0m);
[e] in NaCl (0.1m);
[
30]
[34]
Me
4
NCl (0.1m);
[g] the ionic strength was not controlled (258C); [h] in KCl some Ln(3) and Ln(6) complexes, assuming forma-
[
35]
(
0.1m); [i] the value refers to log b
2
, since this ligand has only two protonation con-
tion of only 1:1 complexes, are listed in Table 2.
The standard deviations associated with these con-
stants significantly decreased when the formation of
monoprotonated species (LnLH) was also taken into consid-
eration in the calculations. This suggests that protonation of
the complexes also occurs under these conditions. The Ce(6)
system was an exception, as the inclusion of the protonated
species in the equilibrium model for this complex resulted in
a significant increase in the standard deviation of fitted pa-
rameters.
stants.
two amide derivatives, DO3A-monopropylamide (5) and
DOTA-tetramethylamide (7). The trends found for the pro-
tonation constants of these ligands are similar, except for
DOTA-tetramethylamide (7; Scheme 2). For DOTA and
Table 2. Thermodynamic stability constants and protonation constants of
lanthanide complexes of ligands 3–7, 258C.
III
[a]
[c]
[f]
[a]
[h]
Ln
Eq.
3
4
5
6
7
[
e]
e]
d]
logKLn·L
logKLnL·H
logKLn·L
logKLnL·H
logKLn·L
logKLnL·H
19.2
–
A
H
U
G
R
N
U
G
–
–
19.26(4)
2.65(5)
12.68
–
13.54
–
13.91
–
III
Ce
[
b]
[d]
20.93(9) 24.67, 25.3
2.93(4) 2.3
21.51(9) 25.41, 25.0
20.1 21.29(1)
III
Gd
–
–
–
2.57(1)
21.83(3)
2.33(4)
[g]
III
Lu
2.44(9)
+
[
[
a] This work in KCl (1.0m); [b] logKLnL·H =[LnHL]/
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
[LnL][H ];
III
c] refs. [3637]; [d] data correspond to the La complex; [e] refs. [3637];
[f] in NaCl (0.1m); [g] data correspond to the Yb complex; [h] in KCl
[
26]
III
Scheme 2. The structures of DOTA (4), a DOTA-tetramethylamide (7)
and some DO3A-monoamide derivatives (5, 6 and 8) discussed in this
work.
[
35]
(1.0m).
III
DO3A-monoamide derivatives (3 and 6), the first two pro-
tonation constants, which have been traced to protonation
at ring nitrogen atoms positioned trans to one another, are
As expected, the stabilities of all Ln complexes increase
III
with decreasing lanthanide ion size, following the Ce
<
III
III
[37]
Gd <Lu order (Table 2).
The stabilities of all Ln(3)
H
relatively high, whereas the remaining logKi values (re-
complexes were similar to the corresponding Ln(6) com-
plexes, as expected for similar DO3A-monoamide struc-
tures. This indicates that incorporation of the elongated ni-
troimidazole-containing pendant arm does little to alter
complex stability. However, these complexes are about four
flecting protonation of the carboxylates) are much lower.
For the monoamide ligands studied here (3 and 6) five pro-
tonation steps were observed in the pH range of 2–12. The
total basicity of these ligands (expressed as the sum of the
protonation constants, logb ) is lower than that of DOTA,
reflecting the lower basicity of the macrocyclic N atoms.
orders of magnitude less stable than the corresponding Ln-
4
[28,29]
À
A( DOTA) complexes due to the substitution of a carboxylate
H
U
G
R
N
U
G
The protonation constants of 6 as determined previously by
pH-potentiometry in 0.1m Me NCl (logb =27.94) were
in DOTA for the weaker amide donor atom in 3 and 6. This
is also reflected in the total basicity of 3 and 6 compared to
DOTA. Further substitution of acetate arms for amides re-
sulted in even lower stabilities as illustrated by the stability
4
4
found to be somewhat higher than the values measured here
[30]
in 1.0m KCl (logb =26.54). This can be attributed to the
4
III
formation of a weak potassium complex in the presence of
of the Ln complexes of 7. It is worth noting that the stabil-
I
1
.0m K ions (a similar decrease in basicity was observed for
ity constant values measured in the presence of KCl repre-
sent a lower limit since these calculations assume no signifi-
DOTA when the protonation constants were measured in
[31]
I
the presence of KCl versus Me NCl).
Interestingly, the
cant interaction between the ligands and K ions, which is
4
first protonation constant of 3 is slightly lower (0.4 logK
units) than that of 6, presumably reflecting the elongated
side arm on the amide. A similar decrease in basicity was
also observed for some alkyl-EDTA derivatives for which
likely to be an invalid assumption. These results indicate
that DO3A-monobutylamide (6) serves as a good model for
the nitroimidazole ligand, 3, and other similar DOTA- or
DO3A-bioconjugate systems.
Chem. Eur. J. 2012, 00, 0 – 0
ꢄ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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A. D. Sherry, G. Tircsꢁ et al.
Relaxometric studies: MRI contrast agents are typically
characterized by a T proton relaxivity (r ) value. The relax-
as the bound water lifetime approaches the slow exchange
condition (T1M ꢀt ), the relaxivity becomes exchange limit-
1
1p
M
[43,44]
ivity (r ) of low molecular weight GdL that has rapid water
ed and its temperature dependence is less pronounced.
1p
exchange kinetics is dominated by the inner-sphere contri-
is
17
bution (r1p ). The Solomon–Bloembergen–Morgan (SBM)
Variable temperature O NMR spectroscopy: The water ex-
change dynamics in Gd(3) and Gd(6) were studied by varia-
is
theory of relaxivity predicts that r1p is dependent on several
1
7
parameters including the number of inner-sphere water mol-
ecules (q), the longitudinal relaxation time of the protons of
ble temperature O NMR measurements. The temperature
dependent profiles of the water O NMR transverse relaxa-
1
7
the water molecule(s) in the inner coordination sphere
tion rates for Gd(3) and Gd(6) (Figure 1) are similar to
H
III
(
T1M ), the residence time of the inner-sphere water mole-
those reported previously for Gd complexes of other small
[41]
cule(s) (t ) and the tumbling rate of the paramagnetic com-
molecular weight complexes, such as Gd(7).
The maxi-
M
[38]
plex in solution (t , rotational correlation time). At clini-
mum in the R2p versus temperature curves for Gd(3) and
Gd(6) were observed at about the same temperature as that
R
cally relevant fields (1.5–3 T), the relaxivity is largely deter-
mined by tM and t . These constants can be obtained by fit-
reported previously for Gd(8) whereas the maximum ob-
R
1
7
1
À
ting of variable temperature O NMR (t ) and H nuclear
served for Gd
A
H
U
G
E
N
N
(DOTA) is shifted to lower temperatures, re-
M
[
38–40]
[41,42]
magnetic relaxation dispersion (NMRD) data (t ).
At
flecting faster water exchange.
The residence time (tM)
R
2
0 MHz, 258C and pH 7, the relaxivity (r ) of Gd(3) and
of the inner-sphere water molecule was determined by fit-
1
p
À1 À1
[45,44]
Gd(6) were 6.38
tively. The r1p value of Gd(3) is about 30% higher than that
of Gd(6), reflecting its higher molecular weight (larger size)
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(Æ0.04) and 5.05
A
H
U
G
R
N
U
G
ting these data to Swift–Connick theory.
The value
found here for Gd(3) (0.71 ms; Table 3) is about 50% of that
found for Gd(8) (1.3 ms) and about four-times longer than
À
[38,41]
and consequently longer rotational correlation time (t ).
that found for Gd ACHTUNGERTUNNNG( DOTA) (t =0.23 ms at 298 K).
M
R
The relaxivity of Gd(6) is nearly the same as that of Gd(8)
These values are consistent with the empirical observation
that substitution of one carboxylate for an amide results in
an approximately three- to fourfold decrease in the metal
bound water exchange rate in Ln complexes of polyamino-
À1 À1
(
r =5.0 mm s , 258C, 20 MHz), a Gd-complex of similar
1p
[41]
molecular weight and size.
[46]
Temperature dependence of relaxivity: The relaxivities of
Gd(3) and Gd(6) were also measured at 378C (20 MHz)
polycarboxylate-type ligands.
The relatively long water
residence times suggest that the water exchange in these
[46]
and found to be only slightly lower (5.25
A
H
U
G
R
N
U
G
complexes likely occur through a dissociative mechanism.
À1 À1
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(Æ0.02) mm s , respectively) than the values measured at
À
258C. The relaxivity of Gd
A
H
U
G
E
N
N
(DOTA) has a similar tempera-
Magnetic field dependence: The magnetic field dependence
ture dependence at this field, decreasing from 4.7 to
of the proton relaxation rates (NMRD profiles) was also
À1 À1
3.6 mm
s
as the temperature
is increased from 25 to
[41,42]
378C.
This behavior is ex-
pected for fast inner-sphere
water exchange systems. It has
been shown that the relaxivity
of low-molecular-weight Gd-
polyaminopolycarboxylate com-
plexes the bound water life-
times of which fall into the fast
exchange regime (T1M @t ) de-
M
creases exponentially with in-
creasing temperature due to
a decrease of the contribution
is
of both r1p (the residence time
of the bound water molecule
os
decreases) and r1p (the diffu-
sion rate of the water molecules
increases on the surface of the
complex). This has been ob-
III
served for the Gd complexes
of DOTA-like ligands that have
two inner-sphere water mole-
cules (q=2; DO3A, PCTA)
and consequently fast water ex-
1
7
Figure 1. Top row: temperature dependence of the transverse water O NMR relaxation rates at 14.1 T and
pH 7 for 21 and 19 mm solutions of Gd(3) (left) and Gd(6) (right), respectively. Bottom row: NMRD profiles
of the 1 mm solutions of Gd(3) (left) and Gd(6) (right) at pH 7. The solid and dashed curves in the lower part
However, represent the inner- and outer-sphere contributions to the overall relaxivity, respectively.
[27,41,42]
change rates.
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4
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A Hypoxia Sensitive MRI Probe
FULL PAPER
Table 3. Relaxation parameters calculated from the simultaneous fitting
of the temperature dependence of the water O transverse relaxation
rate and the NMRD profiles of Gd(3) and Gd(6).
longitudinal relaxation rate of which is dominated by the
outer-sphere relaxation due to the very slow exchange be-
tween the inner-sphere water molecule and the bulk
1
7
[
a]
Gd(3)
Gd(6)
Gd(8)
[49]
water.
2
98
À1 À1
r
q
1p
[mm
s
]
6.38Æ0.04
5.05Æ0.02
5.0
1
1
1
MRI of cells: Cellular uptake of Gd(3) (or Gd(6) as control)
under hypoxic versus normoxic conditions was first exam-
ined by using 9L rat glioma cells as an in vitro model. This
cell line has been extensively used in the past as a hypoxia
model in radiosensitivity studies under low oxygen condi-
2
À2
19
[b]
[b]
D [s ꢆ10 ]
1.9Æ0.1
1.0Æ0.1
1.4
40
70
1.3
19
40
2
98
[b]
[b]
t
t
t
v
[ps]
[ps]
[ms]
58Æ7
34Æ4
2
98
[b]
[b]
R
141Æ3
86Æ2
2
98
[c]
[c]
M
0.71Æ0.09
0.66Æ0.05
À1
[c]
[c]
DH
DH
M
[kJmol
[kJmol
]
]
28Æ4
40Æ3
À1
[c]
[c]
V
25Æ10
28Æ10
[52–54]
tions.
In our experiments, hypoxia was induced in 9L
[
a] Ref. [41]; [b] best-fit parameters obtained from the analysis of the
cells by exposing a plate of cells to nitrogen gas at a rate of
NMRD profile by using the standard value for the average distance be-
tween the metal and protons of the inner-sphere water molecule (a=
À1
1
Lmin for 2 h at room temperature prior to the addition
À5
2
À1
of the complex. After this induction phase, the cells were in-
cubated with either 5 mm Gd(3) or Gd(6) for 2 h under
a continuous flow of nitrogen gas. The control consisted of
the same cells exposed to the same concentration of each
agent but by using a constant flow of air rather than nitro-
gen. In addition, a negative control was prepared in which
cells were incubated with PBS buffer alone (no gadolinium).
After the incubation period, the samples were washed with
PBS, detached from the culture plates with trypsin, centri-
3
.8 ꢇ), and 2.24ꢆ10 cm s for the self-diffusion coefficient of water
[
43,50]
(
D);
[c] best-fit parameters obtained from the analysis of the temper-
ature dependence of O NMR transverse relaxation rate in 21 and
1
7
3
+ 17
1
9 mm solutions of Gd(3) and Gd(6), respectively, assuming a Gd – O
6 III 17
À1
scalar coupling constant of À3.8ꢆ10 rads and a Gd – O distance of
.5 ꢇ.
[
44,51]
2
measured for Gd(3) and Gd(6) at 258C and pH 7 (Figure 1).
Among other relaxation parameters, the rotational correla-
tion time (t ) can be determined from the magnetic field
fuged, and imaged immediately at 4.7 T. The T -weighted
R
1
dependence of the longitudinal relaxation rate of the com-
plex.
images and T maps for three samples: 1) negative control
(C), 2) normoxic incubation (N), and 3) hypoxic incubation
(H) of each complex are shown in Figure 2.
1
In a typical NMRD experiment, a field-cycling relaxome-
ter is used to measure the longitudinal relaxation rates of
the solvent protons in the presence of the paramagnetic
complex over a continuum of magnetic fields that range
from 0.01 MHz up to approximately 70 MHz. Normally, tM
is independently determined by variable temperature
17
O NMR spectroscopy, whereas the inner-sphere relaxivity
parameters are obtained by fitting the NMRD profiles to
the Solomon–Bloembergen–Morgan equations and the
outer-sphere parameters are determined by fitting the data
[47,48]
to Freedꢅs equations.
The solid curves through the ex-
perimental data points (Figure 1) represent the best fit cal-
culated values obtained by use of the relaxation parameters
summarized in Table 3. The NMRD profile of both com-
plexes show a single dispersion centered near 4 MHz with
two plateaus in the low and high magnetic field regions.
(
(
The rotational correlation times of the complexes Gd(6)
t =86 ps) and Gd(8) (t =70 ps) are similar, whereas the
R
R
tR value of the complex Gd(3) (141 ps) is about twice as
[41]
high as that of Gd(8).) Obviously, the long pendant arm
in Gd(3) slows molecular rotation in this complex resulting
in a higher relaxivity. The inner- and outer-sphere contribu-
tion to the r1p relaxivity of Gd(3) and Gd(6) could also be
estimated from the fitting of the NMRD profile (Figure 1).
is
At 258C and 20 MHz, the inner-sphere relaxivity (r ) of
1
p
À1 À1
Gd(3) (4.0 mm s ) is almost twice as high as that of Gd(6)
À1 À1
(
2.4 mm s ) due to its slower tumbling rate. On the other
Figure 2. In vitro MR imaging of 9L rat glioma cells after exposure to
hand, as expected, the contribution of the outer-sphere
either Gd(3) or Gd(6) (top). The T
1 R
-weighted images (T =300 msec;
os
T =10 msec) and T maps of negative control (C), normoxic (N) and hy-
E
1
water molecules to the overall relaxivity (r ) is compara-
1
p
À1 À1
1
poxic (H) cells. Relaxation rates (R ) for the packed cell layers (bottom);
ble (2.5 mm s ) for both complexes. This value is also in
*
=p<0.05 compared to negative control (C); **=p<0.05 compared to
good agreement with the r1p value of Gd(7) (r =
1p
normoxic sample (N). A color version of Figure 2 is included in the Sup-
porting Information.
À1 À1
2
.5 mm s , 258C, 20 MHz), a complex the water proton
Chem. Eur. J. 2012, 00, 0 – 0
ꢄ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
5
&
ÞÞ
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A. D. Sherry, G. Tircsꢁ et al.
If the nitroimidazole moiety in Gd(3) undergoes reduc-
tion and is trapped in hypoxic cells, then the accumulation
of Gd(3) should result in brighter proton images as a result
3 compared to DOTA. The r1p relaxivity, residence time of
the metal bound water molecule (t ) and rotational correla-
M
tion time (t ) of Gd(3) was determined by relaxivity mea-
R
1
7
of the shortening of T of the bulk water by the intracellular
ACHTUNGTRENNUNsG urements, variable temperature O NMR spectroscopy and
1
agent. As seen from the T -weighted images in Figure 2, the
hypoxic sample (H) has higher signal intensity when com-
pared to the other two samples, whereas the normoxic and
proton nuclear magnetic relaxation dispersion (NMRD)
1
À1 À1
studies, and the data (r =6.38 mm s , t =0.71 ms, t =
1
p
M
R
III
141 ps) are in agreement with those obtained for other Gd
negative control samples are indistinguishable. The T maps
also show that cells incubated with Gd(3) under normoxic
complexes of similar size. In vitro MRI experiments with hy-
poxic 9L rat glioma cells revealed that Gd(3) is selectively
trapped in hypoxic cells as evidenced by a nearly twofold
contrast enhancement in the images of cells compared to
normoxic cells. This suggests that this nitroimidazole conju-
gate might be suitable for assessment of hypoxia, in vivo.
Preliminary MR imaging of tumor bearing rats indicates
1
conditions have a somewhat shorter T than the negative
1
control cells. The average T values for the negative con-
1
trols, the normoxic and hypoxic incubation samples were
1
.6
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(Æ0.2), 1.4
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(Æ0.1), and 0.8(Æ0.1) s, respectively. This illus-
A
H
U
G
R
N
U
G
trates that Gd(3) enters cells by passive diffusion in both
cell samples (normoxia and hypoxia) and a small amount of
complex likely gets trapped even in normoxic cells. Never-
[55]
that Gd(3) indeed accumulates largely in hypoxic tissues.
theless, the hypoxic cell pellet displays the shortest T of the
1
three samples. Converting the values to relaxation rates
Experimental Section
À1
(
0.64
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(Æ0.09), 0.74
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(Æ0.14) and 1.31
A
H
U
G
R
N
U
G
highlights the differences in R .
1
p
Abbreviations: MRI: magnetic resonance imaging; PET: positron emis-
sion tomography; SPECT: single photon emission computed tomography;
L: ligand; DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid; DO3A: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; PCTA:
pyclen triacetic acid; NMRD: nuclear magnetic resonance dispersion;
ROI: region of interest.
Subtraction of the R value for the negative control (cells
1
À1
only, no Gd(3)), yields R1p values of 0.10 and 0.67 s for the
samples incubated with Gd(3) under normoxic and hypoxic
conditions, respectively. Thus, the hypoxic cells accumulate
approximately sevenfold more Gd(3) compared to normoxic
General: Details of all synthesis procedures, characterization of all inter-
cells. In comparison, the distribution of T values found for
III
1
mediates, preparation of the Gd complexes, pH potentiometric titra-
normoxic and hypoxic cells incubated with Gd(6) are signifi-
cantly different (p<0.05) from the negative control but not
from each other. The R values of for the samples are 0.45
and 0.56 s under normoxic and hypoxic conditions, respec-
tively. Although the two complexes (Gd(3) and Gd(6))
appear to accumulate to about the same extent in hypoxic
cells as the nitroimidazole derivative, only Gd(3) shows hy-
poxia selectivity. The higher uptake of Gd(6) indicates that
its uptake and retention is mediated by an oxygen-indepen-
tions of ligands and complexes are reported in the Supporting Informa-
tion.
1
Relaxivity and NMRD measurements: The T values were recorded at
1
p
À1
20 MHz (0.47 T), 258C and 378C by using a Stelar Spinmaster spectrom-
eter (Stelar, Italy) and a Maran Ultra relaxometer (Oxford Instruments,
UK) at 23 MHz and 378C. Longitudinal relaxation times were measured
by using the inversion-recovery pulse sequence (1808-t-908). The T1 re-
laxivities were determined by the linear regression analysis of the water
proton relaxation rates in solutions ranging in concentration from 0.2 to
2
1 mm, HEPES buffer (50 mm), in triplicate. The NMRD profiles were
recorded with samples (1 mm) over a continuum of magnetic field
strengths from 0.00024 to 0.47 T (corresponding to 0.01–20 MHz proton
Larmor Frequency) at 258C by using a Stelar field-cycling relaxometer
under computer control with an absolute uncertainty of 1%. Relaxation
data covering the range 0.47 T (20 MHz) to 1.7 T (70 MHz) were collect-
ed by using a Stelar Spinmaster spectrometer operating at variable fields.
ACHTUNGTRENNUNGd ent factor other than just passive diffusion. The higher
uptake of Gd(6) could reflect its lower molecular weight
and/or higher lipophilicity. Whatever the exact reasons for
the nonselective uptake of Gd(6), the important fact is that
this complex does not show hypoxia selectivity, whereas
Gd(3) exhibits selective hypoxia-mediated retention.
1
7
17
Variable temperature O NMR measurements: O NMR linewidth data
were collected by using a Bruker DRX 600 NMR (14.1 T) spectrometer
equipped with a 5 mm probe and a Bruker VT-1000 thermocontroller.
NMR data were acquired by using a spectral width of 10000 Hz, a 908
pulse (7 ms), an acquisition time of 10 ms, 1000 scans and no sample spin-
Conclusion
1
7
ning. Water enriched in O to 2.6% (Yeda, Israel) was used. The ob-
O
served transverse relaxation rates (R 2obs) were calculated from the signal
DO3A-monoamide conjugate of 2-nitroimidazole (ligand 3)
O
width at half-height (Dn1/2): R 2obs =pDn1/2. The paramagnetic contribu-
III
17
O
was synthesized and its complexes with Gd and other lan-
tion to the water O transverse relaxation rate (R 2p) was calculated
O
O
from the R 2obs values according to the following equation: R 2p
=
thanide ions were characterized. As expected, the protona-
tion constants of this ligand and stability of the resulting lan-
thanide ion complexes are quite comparable to published
values for similar compounds. The ligand has a total of five
protonation steps in the pH range of 2–12; two of these are
assigned to the protonation of two macrocyclic nitrogen
atoms. The stability of the Ln(3) complexes ranges from
O
O
O
17
R
2obsÀR 2d, where R 2d is the water O transverse relaxation rate in the
absence of a GdL complex. The concentrations of Gd(3) and Gd(6) used
17
for O NMR measurements were 21 and 19 mm, respectively.
In vitro cell experiments: Rat 9L glioma cells were grown to confluence
in Dulbeccoꢅs Modified Eagle Medium (DMEM) supplemented with
fetal bovine serum (10%), l-glutamine (1%) and penicillin/streptomycin
(
(
1%). The growth medium was replaced with un-supplemented DMEM
8 mL) prior to the uptake experiment. One plate (100 mm i.d.) of cells
III
III
1
9.24 to 21.51 for Ce to Lu , about four orders of magni-
À1
was made hypoxic by being exposed to nitrogen gas (1 Lmin ) in an en-
closed chamber for 2 h at room temperature and two other plates were
exposed to air. PBS (1 mL) was then added to one of the two normoxic
À
tude lower than that of the corresponding Ln
A( DOTA) com-
H
U
G
R
N
N
plexes. This is in accordance with the lower total basicity of
&
6
&
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These are not the final page numbers!

A Hypoxia Sensitive MRI Probe
FULL PAPER
samples (as negative control), and 1 mL of Gd(3) (45 mm) was added to
the remaining two samples (one normoxic and one hypoxic) to yield
a final concentration of 5 mm. The same procedure was followed to pre-
pare three other samples (control, normoxic and hypoxic) for assessing
cell uptake of Gd(6). These samples were incubated for 2 h at room tem-
perature (negative control and reference samples in air, hypoxic sample
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A Hypoxia Sensitive MRI Probe
FULL PAPER
Imaging Agents
F. A. Rojas-Quijano, G. Tircsꢀ,*
E. Tircsꢀnꢁ Benyꢀ, Z. Baranyai,
H. Tran Hoang, F. K. Kꢂlmꢂn,
P. K. Gulaka, V. D. Kodibagkar,
S. Aime, Z. Kovꢂcs,
Hunting hypoxic cells: DO3A-mono-
ACHTUNGTRENNUNGa mide ligand with 2-nitroimidazole
In vitro MRI experiments revealed
that the conjugate might be suitable
for assessment of hypoxia in vivo as
the agent was selectively trapped in
hypoxic (9L rat glioma) cells.
A. D. Sherry* ................. &&&& — &&&&
moiety was synthesized with an aim to
III
Synthesis and Characterization of
a Hypoxia-Sensitive MRI Probe
complex the Gd ion as well as to
target and visualize hypoxic cells by
using the MRI technique (see figure).
Chem. Eur. J. 2012, 00, 0 – 0
ꢄ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!