Activatable magnetic resonance imaging agents for myeloperoxidase sensing: Mechanism of activation, stability, and toxicity

Article abstract of DOI:10.1021/ja905274f

Myeloperoxidase (MPO) is increasingly being recognized as an important factor in many inflammatory diseases, particularly cardiovascular and neurological diseases. MPO-specific imaging agents would thus be highly useful to diagnose early disease, monitor disease progression, and quantify treatment effects. This study reports in vitro and in vivo characterizations of the mechanism of interaction between MPO and paramagnetic enzyme substrates based on physical and biological measurements. We show that these agents are activated through a radical mechanism, which can combine to form oligomers and, in the presence of tyrosine-containing peptide, bind to proteins. We further identified two new imaging agents, which represent the near extremes in either oligomerization (mono-5HT-DTPA-Gd) or proteinbinding in their activation mechanism (bis-o-dianisidine-DTPA-Gd). On the other hand, we found that the agent bis-5HT-DTPA-Gd utilizes both mechanisms when activated. These properties yield distinct in vivo pharmacokinetics profiles for each of these agents that may be exploited for different applications. Specificity studies show that only MPO, but not eosinophil peroxidase, can highly activate these agents, and that MPO activity as low as 0.005 U/mg of tissue can be detected. Gd kinetic lability and cytotoxicity studies further confirm stability of the Gd ion and low toxicity for the 5HT-based agents, suggesting that these agents are suitable for translational in vivo studies.

Full text of DOI:10.1021/ja905274f

Published on Web 12/07/2009
Activatable Magnetic Resonance Imaging Agents for
Myeloperoxidase Sensing: Mechanism of Activation, Stability,
and Toxicity
Elisenda Rodr´ıguez,† Mark Nilges,‡ Ralph Weissleder,†,§ and John W. Chen*,†,§
Center for Molecular Imaging Research, Department of Radiology, Massachusetts General
Hospital, Boston, Massachusetts 02114, Illinois EPR Research Center, UniVersity of Illinois,
Urbana, Illinois 61801, and Center for Systems Biology, Massachusetts General Hospital,
Boston, Massachusetts 02114
Received June 30, 2009; E-mail: jwchen@mgh.harvard.edu
Abstract: Myeloperoxidase (MPO) is increasingly being recognized as an important factor in many
inflammatory diseases, particularly cardiovascular and neurological diseases. MPO-specific imaging agents
would thus be highly useful to diagnose early disease, monitor disease progression, and quantify treatment
effects. This study reports in vitro and in vivo characterizations of the mechanism of interaction between
MPO and paramagnetic enzyme substrates based on physical and biological measurements. We show
that these agents are activated through a radical mechanism, which can combine to form oligomers and,
in the presence of tyrosine-containing peptide, bind to proteins. We further identified two new imaging
agents, which represent the near extremes in either oligomerization (mono-5HT-DTPA-Gd) or protein-
binding in their activation mechanism (bis-o-dianisidine-DTPA-Gd). On the other hand, we found that the
agent bis-5HT-DTPA-Gd utilizes both mechanisms when activated. These properties yield distinct in vivo
pharmacokinetics profiles for each of these agents that may be exploited for different applications. Specificity
studies show that only MPO, but not eosinophil peroxidase, can highly activate these agents, and that
MPO activity as low as 0.005 U/mg of tissue can be detected. Gd kinetic lability and cytotoxicity studies
further confirm stability of the Gd ion and low toxicity for the 5HT-based agents, suggesting that these
agents are suitable for translational in vivo studies.
cancer,10 Parkinson’s disease,11 Alzheimer’s disease,6 and
multiple sclerosis.12,13 Clinical trials have shown that MPO
Introduction
Myeloperoxidase (MPO) is a key enzyme in inflammation.1
It is the most abundant protein of the azurophilic granules of
neutrophils1 and is also found in monocytes,2 macrophages,3
microglia,4 Kupffer cells,5 and neurons.6 MPO contributes to
the host immune defense system by generating potent bacteri-
cidal oxidants. Besides its physiological role in host defense,
MPO and its products have been found and implicated in
numerous diseases including atherosclerosis,7 vasculitis,8 stroke,9
levels can be used to predict patient outcome in myocardial
infarction14 and in apparently normal patients,15 and certain
genotypes in the MPO polymorphism are associated with
increased disease severity.16 MPO-deficient individuals have
been found to be less susceptible to cardiovascular diseases.17
Therefore, MPO is emerging to be an important biomarker for
many diseases.
(10) Trush, A.; Esterline, R. L.; Mallet, W. G.; Mosebrook, D. R.; Twerdok,
L. E. AdV. Exp. Med. Biol. 1991, 283, 399–401.
† Department of Radiology, Massachusetts General Hospital.
‡ University of Illinois.
(11) Choi, D. K.; Pennathur, S.; Perier, C.; Tieu, K.; Teismann, P.; Wu,
D. C.; Jackson-Lewis, V.; Vila, M.; Vonsattel, J. P.; Heinecke, J. W.;
Przedborski, S. J. Neurosci. 2005, 25, 6594–600.
§ Center for Systems Biology, Massachusetts General Hospital.
(1) Klebanoff, S. J. J. Leukoc. Biol. 2005, 77, 598–625.
(2) Nichols, B. A.; Bainton, D. F. Lab InVest. 1973, 29, 27–40.
(3) Sugiyama, S.; Okada, Y.; Sukhova, G. K.; Virmani, R.; Heinecke,
J. W.; Libby, P. Am. J. Pathol. 2001, 158, 879–91.
(4) Gray, E.; Thomas, T. L.; Betmouni, S.; Scolding, N.; Love, S. Brain
Pathol. 2008, 18, 86–95.
(12) Hoy, A.; Leininger-Muller, B.; Kutter, D.; Siest, G.; Visvikis, S. Clin.
Chem. Lab. Med. 2002, 40, 2–8.
(13) Nagra, R. M.; Becher, B.; Tourtellotte, W. W.; Antel, J. P.; Gold, D.;
Paladino, T.; Smith, R. A.; Nelson, J. R.; Reynolds, W. F. J. Neu-
roimmunol. 1997, 78, 97–107.
(5) Brown, K. E.; Brunt, E. M.; Heinecke, J. W. Am. J. Pathol. 2001,
159, 2081–8.
(14) Brennan, M. L.; Penn, M. S.; Van Lente, F.; Nambi, V.; Shishehbor,
M. H.; Aviles, R. J.; Goormastic, M.; Pepoy, M. L.; McErlean, E. S.;
Topol, E. J.; Nissen, S. E.; Hazen, S. L. N. Engl. J. Med. 2003, 349,
1595–604.
(6) Green, P. S.; Mendez, A. J.; Jacob, J. S.; Crowley, J. R.; Growdon,
W.; Hyman, B. T.; Heinecke, J. W. J. Neurochem. 2004, 90, 724–33.
(7) Zhang, R.; Brennan, M. L.; Fu, X.; Aviles, R. J.; Pearce, G. L.; Penn,
M. S.; Topol, E. J.; Sprecher, D. L.; Hazen, S. L. JAMA, J. Am. Med.
Assoc. 2001, 286, 2136–42.
(15) Meuwese, M. C.; Stroes, E. S.; Hazen, S. L.; van Miert, J. N.;
Kuivenhoven, J. A.; Schaub, R. G.; Wareham, N. J.; Luben, R.;
Kastelein, J. J.; Khaw, K. T.; Boekholdt, S. M. J. Am. Coll. Cardiol.
2007, 50, 159–65.
(8) Ishida-Okawara, A.; Oharaseki, T.; Takahashi, K.; Hashimoto, Y.;
Aratani, Y.; Koyama, H.; Maeda, N.; Naoe, S.; Suzuki, K. Inflam-
mation 2001, 25, 381–7.
(16) Nikpoor, B.; Turecki, G.; Fournier, C.; Theroux, P.; Rouleau, G. A.
Am. Heart J. 2001, 142, 336–9.
(9) Re, G.; Azzimondi, G.; Lanzarini, C.; Bassein, L.; Vaona, I.; Guarnieri,
C. Eur. J. Emerg. Med. 1997, 4, 5–9.
(17) Kutter, D.; Devaquet, P.; Vanderstocken, G.; Paulus, J. M.; Marchal,
V.; Gothot, A. Acta Haematol. 2000, 104, 10–5.
9
168 J. AM. CHEM. SOC. 2010, 132, 168–177
10.1021/ja905274f  2010 American Chemical Society

Activatable MRI Agents for Myeloperoxidase Sensing
A R T I C L E S
6H), 3.25 (s, 2H), 2.89 (swide, 4H), 2.82 (swide, 4H), 2.57 (m, 4H);
bis-4-aminophenol-DTPA, 7.21 (d, 4H, J ) 8.4 Hz), 6.81 (d, 4H,
J ) 8.4 Hz), 4.01 (s, 4H), 3.85 (m, 8H), 3.45 (m, 4H), 3.37 (m,
4H); bis-o-dianisidine-DTPA, 7.66 (d, 2H, J ) 8 Hz), 7.92 (d, 2H,
J ) 8 Hz), 7.07 (m, 4H), 6.96 (m, 4H), 3.96 (s, 8H), 3.86 (s, 10H),
3.79 (m, 8H), 3.52 (swide, 5H), 3.88 (swide, 5H). MS (see Supporting
Information Figure SI2): m/z expected mono-5HT-DTPA
(C24H33N5O10 + H)+ 552.22, found 552.80; m/z expected bis-5HT-
DTPA (C34H43N7O10 + H)+ 710.31, found 710.32; m/z expected
bis-4-aminophenol-DTPA (C26H33N5O10 + H)+ 576.22, found
We have previously reported a prototype activatable MPO-
specific MR imaging agent, bis-5HT-DTPA-Gd (DTPA )
diethylene triamine pentaacetic acid), and have studied its use
in murine models of myocardial infarction, multiple sclerosis,
and stroke to report on and track MPO activity in vivo.18-20
Further rational optimization and translation to higher animals
and humans of this class of imaging agents require a deeper
understanding of the activation mechanism, interaction with
endogenous biomolecules, and potential stability and toxicity
issues. In this study, we systematically evaluated this class of
agents through chemical modifications with different charac-
teristics and sensitivity to MPO activity. We identified two new
agents that are capable of achieving high degree of activation
in vitro and in vivo, each with a unique profile that may allow
them to be used in different in vitro and in vivo applications.
We also present direct evidence that details the mechanism of
these agents to explain their behavior in vivo. Furthermore, to
better understand the in vivo effects of this class of potentially
clinically useful agents, we assessed both the Gd kinetic lability
and performed cytotoxicity studies.
576.23; and m/z expected bis-o-dianisidine-DTPA (C42H51N7O12
+
H)+ 846.36, found, 846.70.
Synthesis of the Imaging Agents Mono-5HT-DTP-Gd, Bis-
5HT-DTPA-Gd, Bis-4-aminophenol-DTPA-Gd, and Bis-o-di-
anisidine-DTPA-Gd. The synthesis of corresponding Gd3+ imaging
agents was performed in a 5% citric acid solution (pH ) 5.0) at
room temperature. GdCl3 ·6H2O was used in 1.5-fold excess in
comparison to the chelates, and the reaction was stirred for 1 h.
Final products were purified using HPLC with water and aceto-
nitrile, and peaks were detected at 280 nm. The purity of the
resultant imaging agents was measured by analytical HPLC that
showed >96% purity for all imaging agents (see Supporting
Information Figure SI3). MS (see Supporting Information Figure
SI4): m/z expected mono-5HT-DTPA-Gd (C34H38N7O10Gd + H)+
707.22, found 707.33; m/z expected bis-5HT-DTPA-Gd
(C34H40N7O10Gd + H)+ 865.21, found 865.4; m/z expected bis-4-
aminophenol-DTPA-Gd (C26H30N5O10Gd + H)+ 731.22, found
731.6; and m/z expected bis-o-dianisidine-DTPA-Gd (C42H48-
N7O12Gd + H)+ 1001.36, found 1001.50.
Materials and Methods
All of the chemicals required for this work were obtained from
Sigma Chemical Co. unless otherwise stated (St. Louis, MO).
5-Hydroxytryptamine was obtained from Alfa Aesar (Ward Hill,
MA). DTPA-Gd was purchased as Magnevist from Berlex Inc.
(Montville, NJ). Myeloperoxidase and eosinophil peroxidase were
obtained from Lee Biosolutions (St. Louis, MO). RAW 264.7 and
NIH 3T3 cell lines were obtained from American Type Culture
Collection (Manassas, VA). Dulbecco’s modified Eagle’s medium
(DMEM), penicillin/streptomycin, fetal bovine serum (FBS), and
the other materials for culturing of cells were purchased from Fisher
Scientific (Pittsburgh, PA).
ICP-AES Measurements. To compute the relaxivity for each
imaging agent, the Gd3+ concentration was determinated by
inductively coupled plasma atomic emission spectroscopy (ICP-
AES) using a Perkin-Elmer Optima 5300 V (Galbraith Laboratories,
Knoxville, TN). The relaxation rates were then normalized to the
resultant concentrations to obtain the relaxivity.
Cyclic Voltammetry. Cyclic voltammograms (CV) of the base
substrates and the chelates were recorded using an Autolab
PGSTAT30 potentiostat (Utrecht, The Netherlands). A three-
electrode system was employed: (i) the reference electrode (Ag/
AgCl), (ii) the working electrode (Au), and (iii) a counterelectrode
(platinum wire). CV tracings were recorded from 0 to 0.75 V versus
the reference electrode at a scan rate of 50 mV/s. The Britton-
Robinson (B-R) solution at pH 2 was used as electrolyte.
EPR Measurements. Electron paramagnetic resonance (EPR)
measurements were performed at room temperature using a Varian
E-line X-band spectrometer (Varian E-122 spectrometer). 2-Methyl-
2-nitrosopropane (MNP) was used as the spin-trap. The chelates
(5 mM) as well as the MNP (20 mM) were prepared in 50 mM
Na2HPO4, 100 µM diethylenetriaminepentaacetic acid, pH 8. While
avoiding light exposure, the chelate solutions were mixed with MNP
followed by MPO (10 U or 50 µg/mL) and H2O2 (2 µL of H2O2
(3%)) to initiate agent activation.
Synthesis of the Chelates Mono-5HT-hydroxytryptamide-DTPA
(Mono-5HT-DTPA), Bis-5HT-hydroxytryptamide-DTPA (Bis-
5HT-DTPA), Bis-4-aminophenol-DTPA, and Bis-o-phenylenedi-
amide-DTPA (Bis-o-dianisidine-DTPA). The chelates were syn-
thesized using a modified protocol:21 100 mg (0.28 mmol) of
DTPA-bisanhydride was reacted with the corresponding amines in
DMF (4 mL) in the presence of 100 µL (1 mmol) of pyridine. To
get the bis- or the mono-aminosubstituted chelates, the ratio used
between the base substrates (5-hydroxytrytamine (5HT), o-diani-
sidine, and 4-aminophenol) and the DTPA-bisanhydride was 0.25
for the mono- and 2.2 for the bis-substituted chelates. To avoid
amine oxidation, 50 mg (2.8 mmol) of ascorbic acid was added.
The mixture was stirred for 30 min at room temperature. The crude
product was first precipitated with ether, dissolved in water, and
passed through a C18 cartridge with water and acetonitrile mixtures.
The products were purified using HPLC (water/acetonitrile), and
1
peaks were detected at 280 nm. The purity was confirmed by H
NMR. 1H NMR (400 MHz, D2O) (see Supporting Information
Figure SI1): mono-5HT-DTPA, 7.35 (d, 1H, J ) 8.4 Hz), 7.22 (s,
1H), 7.09 (s, 1H), 6.80 (d, 1H, J ) 12 Hz), 3.87 (s, 4H), 3.80 (s,
2H), 3.67 (s, 2H), 3.59 (t, 2H, J ) 5.6 Hz), 3.46 (s, 2H), 3.29 (t,
2H, J ) 6 Hz), 2.93 (m, 6H), 2.88 (m, 2H), 2.84 (m, 2H); bis-
5HT-DTPA, 7.32 (d, 2H, J ) 8.4 Hz), 7.16 (s, 2H), 7.04 (s, 2H),
6.79 (d, 2H, J ) 8.4 Hz), 3.55 (s, 6H), 3.50 (swide, 4H), 3.41 (s,
Simulations of the EPR spectra were performed using the
program FIT22-24 based on the EPRLF engine developed by
Schneider et al.25 and later by Budil et al.24 to simulate motionally
modulated EPR spectra. A two population model was used to
simulate the spectra for mono-5HT-DTPA and bis-5HT-DTPA,
while a one population model was used to simulate the spectra for
bis-o-dianisidine-DTPA. The simulations assumed isotropic g- and
(18) Nahrendorf, M.; Sosnovik, D.; Chen, J. W.; Panizzi, P.; Figueiredo,
J. L.; Aikawa, E.; Libby, P.; Swirski, F. K.; Weissleder, R. Circulation
2008, 117, 1153–60.
(22) Wiener, E. C.; Auteri, F. P.; Chen, J. W.; Brechbiel, M. W.; Gansow,
O. A.; Schneider, D. J.; Clarkson, R. B.; Lauterbur, P. C. J. Am. Chem.
Soc. 1996, 1996, 7774–7782.
(19) Breckwoldt, M. O.; Chen, J. W.; Stangenberg, L.; Aikawa, E.;
Rodriguez, E.; Qiu, S.; Moskowitz, M. A.; Weissleder, R. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 18584–9.
(23) Chen, J. W.; Clarkson, R. B.; Beldford, R. L. J. Phys. Chem. 1996,
100, 8093–8100.
(24) Chen, J. W.; Auteri, F. P.; Budil, D. E.; Beldford, R. L.; Clarkson,
R. B. J. Phys. Chem. 1994, 98, 13452–13459.
(25) Schneider, D. J.; Freed, J. H. A User’s Guide to Slow-Motional ESR
Lineshape Calculations. Biological Magnetic Resonance; Academic
Press: New York, 1989; Vol. 8.
(20) Chen, J. W.; Breckwoldt, M. O.; Aikawa, E.; Chiang, G.; Weissleder,
R. Brain 2008, 131, 1123–33.
(21) Querol, M.; Chen, J. W.; Weissleder, R.; Bogdanov, A., Jr. Org. Lett.
2005, 7, 1719–22.
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A R T I C L E S
Rodr´ıguez et al.
a-values as well as isotropic rotational dynamics, which we believe
were reasonable given that MNP is a small molecule and that at
room temperature the molecules are at or near the fast-motion
regime.
Specific Activity of the Peroxidases. The specific activity of
myeloperoxidase and eosinophil peroxidase was determined by UV/
vis spectrophotometry on a Varian Cary 50 spectrometer (Palo Alto,
CA), using guaiacol, and monitored at 470 nm according to the
method of Klebanoff et al.26
inflamed tissue in a mouse model. 400 µL of a 1:1 mixture of
Matrigel and minimal essential medium (Cambrex, East Rutherford,
NJ) was injected slowly into the thighs of each mouse 1 h prior to
imaging. The right thigh mixture also contained 10-15 U of MPO
and 4 µL of glucose oxidase (1 mg/mL), while the left thigh mixture
contained no enzyme to serve as an internal control. For the MPO
sensitivity experiments, 0.3 mmol/kg of bis-5HT-DTPA-Gd was
injected intravenously to monitor MPO activity from 5 to 40 U
embedded in Matrigel as described above, and the results were
expressed as U of MPO per mg of gel.
Enzyme-Mediated Polymerization. Reactions with MPO were
carried out at room temperature in the presence of 0.5 mM of the
Gd-imaging agents dissolved in phosphate-buffered saline enriched
with 5.5 mM of glucose. T1 relaxation time was measured before
MPO addition using an inversion-recovery pulse sequence on the
Bruker Minispec (Bruker Analytics, North Bellirica, MA) at 0.47
T (20 MHz) at 40 °C. The reaction was initiated by the addition of
an aliquot containing the enzyme (10 U MPO) and 4 µL of glucose
oxidase (GOX) (1 mg/mL) because GOX can better mimic
hydrogen peroxide production in vivo. Glucose can be oxidized
by GOX to glucono-1,5-lactone generating at the same time
hydrogen peroxide. To avoid GOX from competing with MPO
oxidation of the imaging agents, we optimized the GOX concentra-
tion by titration experiments (between 2 and 8 µL @ 1 mg/mL) to
obtain the largest change in T1 before and after MPO was added
(data not shown). After 30 min of enzyme incubation, T1 was
measured again. The degree of activation by MPO was expressed
as the ratio T1 (before MPO)/T1 (after MPO). To test the specificity
of the imaging probes to report myeloperoxidase activity, we
perform the same measurements in the presence of eosinophil
peroxidase instead of MPO.
MALDI-TOF Measurements. MALDI-TOF mass spectrometry
was performed using an Applied Biosystems Voyager DE Pro at
the Tufts University Core Facility (Boston, MA). The imaging
agents (1 mM) were prepared in PBS enriched with 5.5 mM of
glucose, and the polymerization was initiated by the addition of an
aliquot containing the enzyme (10 U MPO) and 4 µL of glucose
oxidase (GOX) (1 mg/mL).
Interactions with Proteins. This experiment was performed to
determine possible binding between the activated imaging agents
and amino acid residues on the surface of proteins. 0.5 mM of Gd
agents was prepared in the presence of a 1 mg/mL polypeptide
rich in Tyr (Poly (Glu, Tyr) (1:1)) or in the presence of a 1 mg/mL
polypeptide without tyrosine (Poly (Glu, Ala) (6:4)). The T1 values
were measured before and after addition of the MPO using the same
conditions described above.
MR Imaging. The MR imaging experiments were performed
on a 1.5 T whole body GE Signa scanner (GE Healthcare,
Waukesha, WI). For all in vivo MR imaging experiments, precon-
trast T2- (TR ) 2000, TE ) 100, ETL ) 8, NEX ) 4, fast spin
echo sequence, acquisition time ) 4:57 min) and T1-weighted
images (at 1.5 T, TR ) 500, TE ) 11, NEX ) 4, spin echo
sequence, acquisition time ) 5:59 min) with fat saturation were
initially performed to locate the embedded gels. For these experi-
ments, matrix size ) 256 × 128, FOV ) 10 × 7.5 cm, slice
thickness ) 1 mm, 22 slices. The images were acquired in the
coronal plane. The MPO imaging agents (0.3 mmol/kg) were
injected intravenously via the tail vein of the mouse. The mouse
was immediately imaged after the injection of the contrast agent
using serial fat saturated T1-weighted sequences (as above for
precontrast imaging) for at least 3 h.
MPO/Matrigel Imaging Experiments (n ) 13). The protocol
for animal experiments was approved by the institutional animal
care committee. Nine-week-old C57BL6 male mice were obtained
from Jackson Laboratories (Bar Harbor, ME). Matrigel (Beckton-
Dikinson, San Jose, CA), a Ewing’s sarcoma basement membrane
matrix, was used to embed human MPO (Lee Biosolutions, St.
Louis, MO). This was chosen to reflect deposits of human MPO in
Image Analysis. For normalized signal intensity measured on
T1-weighted images, mean ( standard deviation (SD) values were
obtained in regions of interest (ROI). Contrast-to-noise ratios (CNR)
were computed for each ROI. Within each animal, the CNR values
were normalized to the maximum value to compute the relative
CNR (rCNR). The analysis was performed using the software
OsiriX v3.5 (http://www.osirix-viewer.com/).
Cell Viability Studies. The evaluation of the possible cytotoxic
effects for the MPO probes was carried out using NIH 3T3 cells.
In parallel to check the possible cytotoxicity of the probes once
activated by the MPO, RAW 264.7 cells were used. RAW 264.7
cells can generate MPO in the presence of E. coli lipopolysacharide
(LPS) (10 µg/mL). Cytotoxicity was evaluated using the 3-(4,5-
dimethylthiazol-2-yl)2,5-diphenyl-tetrazoilum bromide (MTT) re-
duction assay, which measures cell metabolic activity.27 The Gd
agents were dissolved in DMEM plus 10% FBS at the concentra-
tions of 0.025, 0.05, 0.1, 0.25, 0.75, 1, 2.5, and 5 mM. The cells
were placed in a 96-well cell culture plate (1.5 × 104 cells/100
µL) and treated with the Gd agents and the controlled medium for
12 h, then incubated with 0.5 mg/mL MTT for 2 h. A lysing buffer
that dissolves the blue formazan crystals was added to each well,
and the plates were incubated overnight at 37 °C. Optical density
was measured at 570 nm, and values were expressed as a percentage
of control well values (wells containing cell culture medium without
the Gd agents). Oxidative-sensitive dye, 2′7′-dichlorodihydrofluo-
rescein diacetate, DCFH-DA, was used to confirm MPO release
through formation of radical generation after LPS activation.28 For
that, RAW 264.7 cells cultured in 96-wells, previously treated with
10 µg/mL of LPS for 3 and 24 h, were labeled with DCFH-DA for
20 min in the dark. The intensity of fluorescence signal emitted by
2′7′-dichlorofluorescein (DCF) due to oxidation of DCFH by
cellular ROS was detected using a microplate reader Teca Safire2
(Tecan, Mannedorf, Switzerland) (λexcitation ) 485 nm, λemission
530 nm).
)
Kinetic Stability of the Gd Substrates: Transmetallation. This
method reflects the kinetic stability of the Gd3+ complexes when
competing metal ions such as Zn2+ ions, which can displace the
Gd3+ ion from the chelates, are present. For each reaction, 10 µL
of 250 mM of ZnCl2 in water was added to 1 mL of 2.5 mM
imaging agents in pH ) 7 phosphate buffer. We monitored the
evolution of T1 values of the solutions for 3 days. The results are
expressed using two indices: a kinetic index, which is the time
required to reach 80% of the initial R1 value, and a thermodynamic
index, which is the R1(t ) 4320)/R1(t ) 0) value measured after 3
days (4320 min).
Results
Redox Characterization of the MPO Imaging Agents. The
MPO catalytic center consists of a heme group that can undergo
redox reactions described in Scheme 1.
In the presence of H2O2, the native enzyme can be oxidized
to form the redox intermediate compound I, which can transform
halides, particularly Cl-, into hypochlorous acid, HOCl, a potent
antimicrobial oxidant. Compound I is also capable of catalyzing
(27) Hansen, M. B.; Nielsen, S. E.; Berg, K. J. Immunol. Methods 1989,
119, 203–10.
(28) Kimura, K.; Ito, S.; Nagino, M.; Isobe, K. Biochem. Biophys. Res.
Commun. 2008, 372, 272–5.
(26) Klebanoff, S. J.; Waltersdorph, A. M.; Rosen, H. Methods Enzymol.
1984, 105, 399–403.
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Activatable MRI Agents for Myeloperoxidase Sensing
A R T I C L E S
Scheme 1. Myeloperoxidase (MPO) Cycle and Standard Reduction
Potential (E°) for Each Part of the Cyclea
in the presence of MPO, we performed electron paramagnetic
resonance (EPR) spin trapping experiments using the spin trap
2-methyl-2-nitrosopropane (MNP). The EPR spectrum for MNP
is characterized by a low background three-line spectrum
corresponding to the light-induced formation of di-tert-butyl-
nitroxide (17 G) (Figure 2e).30
After the addition of MPO and H2O2 to the chelates, we
observed an increase in the signal intensity for mono-5HT-
DTPA, bis-5HT-DTPA, and bis-o-dianisidine, signifying the
formation and trapping of free radicals (Figure 2a-c). Control
solutions of MNP with MPO and H2O2 but without the substrates
resembled the baseline MNP spectrum (Supporting Information
Figure SI5). A previous EPR study of MPO-tyrosine radical
generation showed an aN of 15.6 G for the MNP-tyrosyl radicals
adduct.30 However, in contrast to the tyrosine EPR spectrum
with a single population of aN ) 15.6 G, two populations were
observed with aN of 15.1 and 17.1 G for the 5HT compounds
(see Figure 2f and g). The aN of 15.1 G for both mono- and
bis-5HT-DTPA confirmed the generation of MNP-5HT radical
adducts after MPO activation. The 17.1 G lines likely resulted
from the decomposition of MNP, which appeared to be
facilitated in the presence of the 5HT groups, because tyrosine
and bis-o-diansidine-DTPA did not exhibit this. Simulations of
the 5HT spectra with a two populations model yielded 49% for
mono-5HT-DTPA and 35% for bis-5HT-DTPA for the 15.1 G
population. For bis-o-dianisidine-DTPA, a single aN of 15.8 G
was observed (Figure 2h). Finally, for bis-4-aminophenol-DTPA,
no significant radical formation was observed (Figure 2d),
corroborating the cyclic voltammetry results that bis-4-amino-
phenol-DTPA did not have a detectable redox property. These
findings show that bis-4-aminophenol-DTPA cannot be oxidized
by MPO and explains the lack of relaxivity change when MPO/
H2O2 was added to bis-4-aminophenol-DTPA-Gd. Therefore,
this agent was not used in the subsequent studies.
In Vitro Relaxometric Characterization of the MPO
Imaging Substrates. In vitro characterization of the ability of
the gadolinium substrates to report MPO activity was performed
using relaxometric methods. In the presence of MPO and
hydrogen peroxide, we have shown that these MPO imaging
agents can dimerize and oligomerize after being activated by
compounds I and II (please also see Supporting Information
Figure SI6).31,32 These changes result in an increase of the agent
size with consequent increase in the T1 relaxivity (Scheme 2).
The mono-5HT-DTPA-Gd complex showed the largest T1
change after activation, demonstrating that this agent has the
highest degree of oligomerization in vitro when activated by
MPO (Table 2) (also see Supporting Information Figure SI6a).
Interestingly, the bis-o-dianisidine-DTPA-Gd agent showed
a small T1 change after MPO addition as compared to the other
two complexes, corroborating the lower degree of the polym-
erization in vitro seen by MALDI-TOF (see Supporting
Information Figure SI6c). Eosinophil peroxidase, EPO, another
human peroxidase found in certain inflammatory conditions, was
also used to test the polymerization of our imaging agents.
Unlike MPO, which causes >1.5-fold T1 relaxivity change in
the agents, the T1 relaxivity barely changed with EPO for the
candidate agents (Table 2). These in vitro findings further
corroborated our previous in vivo findings that no agent
activation was observed in myocardial infarction and stroke
a The efficacy of compound II to complete the cycle is key to MPO
agent design. For an agent to be a substrate instead of an inhibitor for MPO,
Esubstrate needs to be less than the reduction potential of compound II of
0.97 V.
the oxidation of aromatic substrates like tyrosine or 5-hydroxy-
tryptamine through 1e- oxidation to form compound II, which
can itself also oxidize aromatic substrates to recover the native
enzyme. The resultant oxidized substrate can form dimers and
oligomers. Therefore, to systematically assess whether a given
compound is a suitable substrate for MPO, we evaluated the
reduction potential of candidate agents. Based on the standard
reduction potential relative to the MPO intermediates (compound
I/compound II ) 1.35 V; compound II/native state ) 0.97 V),
the recovery of the native enzyme can only take place when
the reduction potential of the substrate is lower than 0.97 V
(Scheme 1).29 Therefore, we sought substrates that have
reduction potentials less than 0.97 V. We identified the base
substrates 4-aminophenol, o-dianisidine, and 5-hydroxytryptamine
as potential MPO substrates that, in addition to having a
reduction potential <0.97 V, also may be conjugated to DTPA.
These substrates demonstrated a significant UV/vis spectral shift
upon the addition of MPO and H2O2, consistent with MPO-
mediated oxidation and oligomerization (data not shown). These
substrates were then conjugated to the chelating agent DTPA
as potential MR imaging agents (Figure 1).
To determine the reduction potential of the chelates, we
performed cyclic voltammetry measurements. The results are
shown in Table 1.
To check if the conjugation of the base substrate to the DTPA
backbone changes their redox properties, Table 1 also shows
the redox properties for the unmodified base substrates. We
found no significant change in the reduction potential upon
conjugation of the substrates to DTPA, except for bis-4-
aminophenol-DTPA, which lost its redox properties once
conjugated to DTPA. In this study, we assume that the redox
properties of the chelates would not change after complexation
to Gd3+
.
EPR Measurements Reveal Radical Generation upon
Agent Activation by MPO. MPO-mediated dimerization and
oligomerization of tyrosine has been found to proceed via free
radical formation and 1e- donation to MPO compounds I and
II.30 However, this has not been confirmed for other types of
MPO substrates such as indoles and o-dianisidine compounds.
To determine if these other substrates also undergo radicalization
(29) Furtmuller, P. G.; Arnhold, J.; Jantschko, W.; Pichler, H.; Obinger,
C. Biochem. Biophys. Res. Commun. 2003, 301, 551–7.
(31) Chen, J. W.; Querol Sans, M.; Bogdanov, A., Jr.; Weissleder, R.
Radiology 2006, 240, 473–81.
(30) McCormick, M. L.; Gaut, J. P.; Lin, T. S.; Britigan, B. E.; Buettner,
G. R.; Heinecke, J. W. J. Biol. Chem. 1998, 273, 32030–7.
(32) Querol, M.; Chen, J. W.; Bogdanov, A. A., Jr. Org. Biomol. Chem.
2006, 4, 1887–95.
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Figure 1. MR imaging agents described in this work, based on the DTPA-Gd backbone conjugated with the base substrates (5-hydroxytryptamine (5HT),
o-dianisidine, and 4-aminophenol). Imaging agents synthesized are: mono-5HT-DTPA-Gd, bis-5HT-DTPA-Gd (a, refs 27, 28), bis-o-dianisidine-DTPA-Gd,
and bis-4-aminophenol-DTPA-Gd. Relaxivity values (mM-1 s-1) for each imaging agent are also shown.
Figure 2. EPR spectra of the radicals generated after MPO-H2O2 activation using the 2-methyl-2-nitrosopropane (MNP) as a spin trap. (a) Mono-5HT-
DTPA, (b) bis-5HT-DTPA, (c) bis-o-dianisidine-DTPA, (d) bis-4-aminophenol-DTPA, (e) MNP only. (f and g) Amplified spectra with simulations to better
show the two populations for mono-5HT-DTPA and bis-5HT-DTPA, and (h) the single population for bis-o-dianisidine. Chelates were prepared in phosphate
solution pH ) 8 and activated using MPO (10 U) and H2O2 (2 µL of 3% H2O2).
Table 1. Reduction Potential (E) Measured from Cyclic Voltammetry at 298 K and pH 2 for the Chelates and for Their Parent Base
Substrates (Bold)
bis-o-
dianisidine-DTPA
bis-4-aminophenol-
DTPA
5HT
mono-5HT-DTPA
bis-5HT-DTPA
o-dianisidine
4-aminophenol
E(V)
0.71
0.62
0.63
0.57
0.65
0.67
not detectable
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Scheme 2. Activation Mechanism of the MR Imaging Agents by the MPO-H2O2 Systema
a Myeloperoxidase, MPO (green bubbles), is localized in the azurophilic granules (blue bubbles) of the neutrophils. The MPO-sensing moiety conjugated
to the gadolinium complex can be oxidized by MPO in the presence of H2O2 to result in radical formation. The radicals can form dimers and oligomers with
other radicals and can bind to other phenolic residues, such as tyrosine, on the protein surface. Both activation effects result in higher T1 relaxivity. The
binding to proteins can further result in prolonged enhancement of the activated agents.
Table 2. In Vitro Assessment of the MR Imaging Agents Using
MR Imaging Reveals Different in Vivo Behavior for the
Agents. To validate the lead agents for in vivo imaging, we
performed MR imaging after the injection of the test imaging
agents in mice, using Matrigel to embed the MPO in the right
thighs of mice. The choice of Matrigel as inflammation model
instead of, for instance, E. coli LPS injected intramuscularly
was due to higher reproducibility of the Matrigel model. All
three agents demonstrated a large increase in enhancement (more
than 2-fold increase in CNR) in the MPO-containing gel (right)
as compared to the control gel (left), consistent with MPO-
mediated activation (Figure 3a). In contrast, in a previous study,
we performed control experiments using the nonspecific DTPA-
Gd that did not demonstrate any increased enhancement in the
MPO-containing gel.31
Relaxometric Measurements (0.47 T)a
T1 ratios before/after MPO
buffer
solution
human
peroxidase
mono-5HT-
DTPA-Gd
bis-5HT-
DTPA-Gd
bis-o-dianisidine-
DTPA-Gd
PBS
PBS
MPO
EPO
MPO
1.8
1.2
1.8 (0%)
1.7
1.2
1.7 (0%)
1.5
1.0
1.5 (0%)
PBS + polypeptide
(Ala-Glu)
PBS + polypeptide
(Tyr-Glu)
MPO
2.2 (22%) 2.7 (59%)
2.1 (40%)
a Eosinophil peroxidase (EPO) was also used to test the specificity of
the agents. Results obtained are expressed as the ratio (T1 before MPO)/
(T1 after MPO). We also repeated the same experiments in the presence
of either poly(Glu,Tyr) or poly (Glu,Ala) polypeptides.
models using MPO-/- mice with the prototype bis-5HT-DTPA-
Gd agent.18,19 These results confirm specificity of the agents
for MPO.
Interestingly, as may be predicted from the in vitro results,
there were substantial differences in the rate of agent activation
and clearance between these agents. The activated mono-5HT-
DTPA-Gd reached peak increased T1-weighted enhancement
during the first hour of imaging and was cleared at nearly the
same rate as the parent, nonactivated agents. For the activated
bis-5HT-DTPA-Gd, the peak occurred at around 90 min after
agent administration, and there was a slower clearance rate as
compared to that of the control site. The activated form of bis-
o-dianisidine-DTPA-Gd, interestingly, did not seem to be
clearing within the experimental time frame of 3 h and continued
to show an increase in enhancement over time.
Activated Agents Interact with Proteins Containing Tyro-
sine. Phenols such as tyrosine have been found to be capable
of cross-linking proteins after oxidation by MPO.31,33 It has
not been shown if other species such as indoles and
o-dianisidine can also cross-link proteins when oxidized by
MPO. Furthermore, it is possible that such cross-linking
ability may be altered by the chemical modifications needed
to conjugate these substrates to a chelating backbone such
as DTPA. In these experiments, we found that the imaging
agents, when activated by MPO, demonstrated a further
increase in relaxivity when exposed to a polypeptide contain-
ing tyrosine residues, confirming that they can cross-link
proteins (Table 2). However, each agent showed different
degrees of relaxivity increase. Both bis agents demonstrated
a large increase in relaxivity (>40%), while the mono-5HT-
DTPA-Gd agent showed a mild increase in relaxivity
(∼20%). In contradistinction, no relaxivity change was
observed when the agents were activated in the presence of
a control peptide that did not contain tyrosine for any of the
three imaging agents, confirming that the cross-linking occurs
between the activated substrates and the tyrosine residues.
To evaluate the sensitivity of the agents for different
concentrations of MPO, we varied the amount of MPO
embedded in the gels and imaged these mice using the same
dose of bis-5HT-DTPA-Gd. Over the range of MPO concentra-
tions we tested, we found that the enhancement for bis-5HT-
DTPA-Gd was linearly proportional to MPO concentration
(Figure 3b) and was able to detect an MPO concentration as
low as 0.005 U/mg of gel. This would allow sensitive detection
of MPO activity at the earliest stages of inflammation (e.g., in
a murine stroke model, the MPO level was 0.15 U/mg 1 day
after stroke induction).19
Kinetic Stability of the Gadolinium Complexes. One method
to assess the stability of the gadolinium complexes is to measure
the degree of transmetallation in the presence of another
(33) Heinecke, J. W. Toxicology 2002, 177, 11–22.
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J. AM. CHEM. SOC. VOL. 132, NO. 1, 2010 173

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Rodr´ıguez et al.
Figure 3. (a) In vivo assessment of the agents mono-5HT-DTPA-Gd, bis-5HT-DTPA-Gd (data taken from ref 31), and bis-o-dianisidine-DTPA-Gd as MPO
activity reporters. The right side (R) contained human MPO and glucose oxidase embedded in Matrigel. The left side (L) contains only Matrigel and
medium. All three agents were able to identify areas of MPO activity. The line graphs illustrate the evolution of contrast-to-noise ratios (CNR) for each
imaging agent in the presence of MPO versus control, demonstrating striking differences in the pharmacokinetics of each activated agent from the nonactivated
parent agent as well as from the other agents. (b) Sensitivity of the bis-5HT-DTPA-Gd agent to report MPO concentrations in vivo.
Table 3. Zinc Transmetallation Kinetic Stability of Gd3+ of the
competing metal.34 Zn2+ is a good competing metal ion because
Imaging Agents in the Presence of Equimolar Zn2+ (2.5 mM)a
it is present in high concentration in blood (55-125 µmol/L)
and because it has an affinity to DTPA similar to that of Gd3+.34
R1(t ) 4320)/R1(t ) 0) t for R1(t)/R1(t ) 0) ) 0.8
Because the displacement of Gd3+ by Zn2+ will result in a
decrease in the T1 relaxation rate (R1) due the precipitation of
the Gd3+ ions in phosphate buffer as Gd(PO4), the effect of the
transmetallation can be evaluated through two parameters: a
kinetic index given by the time required to lose 20% of the
initial R1 value and a thermodynamic index, which is the
percentage of R1 after 3 days as compared to the initial value
(see Supporting Information Figure SI7). From both indices,
the imaging agents modified with 5HT were similar to or better
than that observed for the clinical, conventional agent DTPA-
Gd, especially the bis-version of the agent, suggesting that Gd3+
metal center is not easily displaced by competing Zn2+ ions
(Table 3). However, the bis-o-dianisidine-DTPA-Gd complex
demonstrated substantially lower transmetallation indices, sug-
bis-5HT-DTPA-Gd
mono-5HT-DTPA-Gd
bis-o-dianisidine-DTPA-Gd
DTPA-Gd
0.76
0.48
0.25
0.56
0.09
2866
367
67
250
BMA-DTPA-Gd
50-60b
a Relaxation rate (R1) (s-1) was monitored for 3 days (t reported in
min) at 40 °C. Results are expressed by two indexes: kinetic index
(R1(t)/R1(t ) 0) ) 0.8) and a thermodynamic index (R1(t ) 4320)/R1(t )
0)). b Data taken from ref 34.
gesting the Gd3+ in this complex could be more easily displaced
by the Zn2+ ions.
Cytotoxicity. To evaluate the possible toxic effects of the Gd-
based complexes, we evaluated the cytoxicity in the NIH 3T3
cell line (Figure 4a).27 There was no significant toxicity effect
up to 5 mM for the bis-5HT-DTPA-Gd agent and for the mono-
5HT-DTPA-Gd, well above the expected in vivo concentration.
(34) Laurent, S.; Elst, L. V.; Copoix, F.; Muller, R. N. InVest. Radiol. 2001,
36, 115–22.
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Figure 4. (a) Cell viability of NIH 3T3 cells in the presence of mono-5HT-DTPA-Gd (gray open), bis-5HT-DTPA-Gd (black filled), and bis-o-dianisidine-
DTPA-Gd (gray filled) evaluated using the MTT assay. (b) Cell viability of quiescent RAW cells in the presence of bis-5HT-DTPA-Gd (black filled) was
not significantly altered when the RAW cells were activated with 10 µg/mL of LPS (square filled), evaluated using the MTT assay. Confirmation of RAW
cell activation was confirmed by the detection of ROS species using the DCFH-DA dye after LPS activation (inset).
However, as can be predicted from the zinc transmetallation
study, the bis-o-dianisidine-DTPA-Gd agent demonstrated sub-
stantial toxic effect at 0.3 mM (<80% viable cells), with <50%
viable cells at 5 mM. To further evaluate if the activation of
the agents would cause additional toxicity, we also studied the
toxicity in macrophages (RAW cells) with and without incuba-
tion with LPS to activate the macrophages. Upon stimulation
by LPS, the RAW cells would secrete MPO to activate the MPO
substrates.35 To confirm activation of the macrophages under
this experimental condition, we measured radical generation
using the DCFH-DA dye (inset, Figure 4b). We found no
difference in viability (Figure 4b) between the activated cells
and the nonactivated cells for bis-5HT-DTPA-Gd, consistent
with that the activated MPO imaging agents do not present
additional cytotoxicity effects.
enzyme activity in vitro and in vivo. Furthermore, using a 5HT-
based agent, we showed high sensitivity of the agents for MPO
activity as low as 0.005 U/mg.
We found that one of the chelates, bis-4-aminophenol-DTPA,
despite having a suitable reduction potential prior to DTPA
conjugation, cannot be oxidized by MPO. This was because
the conjugation of the base substrates to DTPA occurs through
an amide bond, which is a strong electron-withdrawing group,
resulting in the subtraction of the electron density from the
aromatic moiety.36 This amide bond is directly attached to the
phenolic group, whereas for the other imaging agents, this
electron-withdrawing group is far enough to not disturb the
electron density of the aromatic moieties. Design of future
optimized imaging agents needs to take into account that
conjugated base substrates (chelates) with strong electron-
withdrawing groups may not be polymerized by MPO because
of the loss of the reduction redox potential.
Discussion
In this study, we showed that MPO oxidation leads to
radicalization of the imaging agents. These intermediate com-
pounds can combine to form dimers and oligomers as well as
cross-link proteins containing tyrosine residues. These MPO-
mediated changes increase the molecular size and consequent
shortening of T1, resulting in >2-fold increased T1-weighted
enhancement on in vivo imaging relative to agents that are not
activated. We have identified that the reduction potential (<0.97
V to allow the recovery of the native form of the MPO) of the
base substrates (5HT, o-dianisidine, and 4-aminophenol) after
conjugation to a chelating backbone is an important indicator
of whether the conjugated substrate could be activated by MPO.
We also demonstrated in this and previous studies that only
MPO, but not EPO, can highly activate these agents to report
We found that mono-5HT-DTPA-Gd demonstrated the high-
est increase in relaxivity in vitro as well as the highest degree
of oligomerization. The extent of oligomerization is likely
related to the accessibility of the probe to the active site of the
enzyme. The conjugation of aromatic phenolic compounds to
DTPA increases steric hindrance that can diminish interactions
with the MPO active site. Thus, the monosubstituted agent, being
smaller, is able to better reach the active site, consistent with
this agent having the highest degree of polymerization (Figure
SI6a). Interestingly, while bis-o-dianisidine-DTPA generated the
highest amount of radicals (Figure 2c), this substrate yielded
very little change in T1 upon activation by MPO in vitro.
Because in vitro the relaxivity increase is mainly due to size
(36) Xu, Y. P.; Huang, G. L.; Yu, Y. T. Biotechnol. Bioeng. 1995, 47,
(35) Cuschieri, J.; Maier, R. V. Antioxid. Redox Signal 2007, 9, 1485–97.
117–9.
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Rodr´ıguez et al.
increase, the larger and floppier conformation of the o-
dianisidine agent may prevent optimal interaction with MPO
to generate larger oligomers. This result confirms that, in
addition to the redox properties, substrate oxidation by MPO is
governed by structural and topological characteristics. The in
vitro efficiency of MPO-mediated activation of the mono-5HT-
DTPA-Gd is verified in vivo (Figure 3a) where mono-5HT-
DTPA-Gd is the agent that is the earliest to peak, consistent
with fast interactions with MPO.
Another contribution that affects in vivo image enhancement
is protein binding. The mono-5HT agent demonstrated the
smallest increase in the T1 ratio in the presence of poly(Glu,
Tyr) as compared to the bis-5HT version, suggesting that a bis-
conformation facilitates protein binding to result in higher
relaxivities, likely by offering an additional moiety for MPO
oxidation. Interestingly, the bis-o-dianisidine-DTPA-Gd agent,
which in the absence of peptides showed the weakest activation
by MPO likely due to its larger size, showed high relative
increase in relaxivity in the presence of poly(Glu, Tyr). This
finding is corroborated in vivo as this agent showed a large
increase in CNR at later time point and did not show substantial
clearance over 3 h, consistent with that most of its in vivo
efficacy is derived from binding of the activated agent to
proteins.
In light of the recent evidence linking nephrogenic systemic
fibrosis (NSF) with the presence of free Gd3+ in renal failure
patients, we chose to evaluate the stability of the agents using
a transmetallation method, which evaluates the kinetic lability
of the Gd3+ ion when other competing metal ions such as Zn2+
are also present at physiological conditions. Our results showed
that the 5HT-based imaging agents were similar to or even better
than DTPA-Gd in Gd kinetic lability. On the other hand, bis-
o-dianisidine-DTPA-Gd complex was found to be very suscep-
tible to transmetallation, with transmetallation indices similar
to Omniscan, the agent most commonly associated with NSF.
This increased Gd kinetic lability may be caused by the
noncoplanar conformation of o-dianisidine, which could allow
increased access of Zn2+ to the chelate, whereas in 5HT the
indolic rings are in a more rigid coplanar conformation,
increasing steric hindrance. The very slow clearance of the
o-dianisidine agent could further increase its in vivo toxicity.
However, as its parent base substrate, o-dianisidine (3,3′-
dimethoxybenzidine), is used in in vitro assay kits to report MPO
activity, bis-o-dianisidine-DTPA-Gd could be used to screen
MPO activity in biological samples in vitro using relaxometric
methods.37,38 On the other hand, for the 5HT-based agents, no
cytotoxicity was identified at doses likely exceeding in vivo
levels of the agent (estimated to be ,1 mM) using either the
parent or the activated agents, suggesting a safe profile for
translation to preclinical and clinical studies. Because these
agents are based on the DTPA backbone, it is expected that
most of the agents will be cleared predominantly by the kidneys
and liver, similar to DTPA-Gd. Indeed, in a biodistribution study
for bis-5HT-DTPA, we found >90% of the radiolabeled agents
to be cleared within 6 h, with the remaining agents found
primarily in the liver, kidneys, spleen, and bowel.31 For activated
agents, in a study on mouse experimental autoimmune encepha-
lomyelitis using bis-5HT-DTPA-Gd, we found no evidence of
residual enhancement 6 h after agent injection in the diseased
organ, suggesting that the binding of activated agents to proteins
is reversible and likely cleaved by proteases that are present
abundantly in inflamed tissues.20
The MPO levels found in human pathological conditions are
significantly higher than the MPO concentrations (∼0.05 U/mg)
used for this work (e.g., in carotid plaques, MPO has been found
to be ∼250 U/mg),38 and should be readily assessed by the
imaging agents described here to detect early, presymptomatic
disease. This, together with the increased signal observed once
these agents are activated, likely will allow much lower doses
to be used in translational studies to further improve the safety
profile. Future optimization of the MPO agents could use a
macrocyclic backbone instead of DTPA to further improve the
Gd kinetic lability. In addition, these chelates are readily
complexed to radioisotopes, for example, 111In, to convert into
nuclear imaging agents that can be used at trace doses.31
Conclusions
We identified two new candidate MPO-sensitive imaging
agents, in addition to our previously reported prototype agent.
Each of these agents possesses a unique activation profile, with
different degrees of oligomerization and protein cross-linking
capabilities. All three agents were efficacious at identifying sites
of MPO activity in vivo. Most of the increased enhancement
observed for the mono-5HT-DTPA-Gd agent is from radical
formation and subsequent oligomerization, while for the bis-
o-dianisidine-DTPA-Gd agent the increased enhancement was
found to be from protein cross-linking, resulting in significantly
delayed clearance of the activated agent. These two agents
represent near extremes in the activation mechanism. For bis-
5HT-DTPA-Gd, the increased enhancement is from both
moderate oligomerization and substantial protein cross-linking,
causing slower clearance of the activated agents. Moreover, we
observed that the activated agents are cleared from mice within
4-6 h20 and less than 24 h in rabbits,39 likely from cleavage
by proteases that are abundant at inflamed sites. The differential
clearance rates of the activated versus nonactivated bis-5HT-
DTPA-Gd agent allow for confirmation of MPO activity at a
slightly delayed imaging time point, when the nonactivated
agents are mostly cleared from the tissues. On the other hand,
mono-5HT-DTPA-Gd may represent an attractive agent in
applications where faster diagnosis is required. As MPO is
implicated in many prevalent diseases such as atherosclerosis,
stroke, and multiple sclerosis, the unique properties identified
in this study for each prototype agent could be exploited for
different in vitro and in vivo environments and applications.
Therefore, future rational development and translation of these
MPO-sensitive and other bioactivatable MR imaging agents40-43
holds promise in bringing biological functional information to
MR imaging.
Acknowledgment. We thank Ben Bautz, Alexy Chudnovsky,
Martin Etzrodt, and Christopher N. LaFratta for experimental
assistance and Brian Rutt for helpful discussions. We also
acknowledge Alexei Bogdanov and Manel Querol for early
(39) Ronald, J. A.; Chen, J. W.; Chen, Y.; Hamilton, A. M.; Rodriguez,
E.; Reynolds, F.; Hegele, R. A.; Rogers, K. A.; Querol, M.; Bogdanov,
A.; WeisslederR.; Rutt, B. K. Circulation 2009, 120, 592–599.
(40) Major, J. L.; Boiteau, R. M.; Meade, T. J. Inorg. Chem. 2008, 47,
10788–95.
(37) Spalteholz, H.; Furtmuller, P. G.; Jakopitsch, C.; Obinger, C.; Schewe,
T.; Sies, H.; Arnhold, J. Biochem. Biophys. Res. Commun. 2008, 371,
810–3.
(41) Major, J. L.; Meade, T. J. Acc. Chem. Res. 2009, 42, 893–903.
(42) Major, J. L.; Parigi, G.; Luchinat, C.; Meade, T. J. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 13881–6.
(38) Daugherty, A.; Dunn, J. L.; Rateri, D. L.; Heinecke, J. W. J. Clin.
InVest. 1994, 94, 437–44.
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Supporting Information Available: 1H NMR and mass spectra
of chelates; HPLC and mass spectra of the imaging agents; EPR
spectra of control solutions; MALDI-TOF mass spectra of
imaging agents after MPO activation; and zinc transmetallation
profiles of the imaging agents. This material is available free
of charge via the Internet at http://pubs.acs.org.
contributions to the development of MPO sensing agents. E.R. was
supported by the EC-Marie Curie grant OIF (Molecular Imaging
39639). This work was supported in part by National Institutes of
Health Grants KO8HL081170 (J.W.C.) and RO1-HL078641
(R.W.).
(43) Delli Castelli, D.; Terreno, E.; Carrera, C.; Giovenzana, G. B.; Mazzon,
R.; Rollet, S.; Visigalli, M.; Aime, S. Inorg. Chem. 2008, 47, 2928–
30.
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