PiB-conjugated, metal-based imaging probes: Multimodal approaches for the visualization of β-amyloid plaques

Article abstract of DOI:10.1021/ml400042w

In an effort toward the visualization of β-amyloid plaques by in vivo imaging techniques, we have conjugated an optimized derivative of the Pittsburgh compound B (PiB), a well-established marker of Aβ plaques, to DO3A-monoamide that is capable of forming

Full text of DOI:10.1021/ml400042w

Letter
pubs.acs.org/acsmedchemlett
PiB-Conjugated, Metal-Based Imaging Probes: Multimodal
Approaches for the Visualization of β‑Amyloid Plaques
Andre F. Martins,†,‡ Jean-Francois Morfin,† Anna Kubíckova,†,§ Vojtech Kubícek,† Frederic Buron,∥
̧
̌ ́ ̌ ̌ ́ ́
́
Franck Suzenet,∥ Milena Salerno,⊥ Adina N. Lazar,# Charles Duyckaerts,# Nicolas Arlicot,¶
¶
,‡
,†
́
Denis Guilloteau, Carlos F. G. C. Geraldes,* and Eva Toth*
́
†Centre de Biophysique Molec
́
ulaire, CNRS, Rue Charles Sadron, 45071 Orlea
́
ns Cedex 2, France
‡Department of Life Sciences, Center of Neurosciences and Cell Biology (CNC), and Coimbra Chemistry Center, University of
Coimbra, Portugal
§Department of Analytical Chemistry, Charles University in Prague, Albertov 2030, 12840 Prague, Czech Republic
∥Institut de Chimie Organique et Analytique, UMR 7311 CNRS/Universite
́
d’Orlea
Paris 13, 74 rue Marcel Cachin, 93017 Bobigny, France
#Centre de Recherche de l’Institut du Cerveau et de la Moelle, CNRS UMR7225, INSERM, UMR975 and UPMC, Ho
pital de la
́ ́
ns, rue de Chartres, 45067 Orleans, France
⊥Laboratoire CSPBAT, CNRS UMR 7244, UFR-SMBH, Universite
́
̂
Pitie-
́
Salpet
̂
rier
̀
e 47, Bd de l’Ho
¶Inserm, U930, Universite
̂
pital 75013 Paris, France
́
Francois Rabelais de Tours, CHRU de Tours, 37044 Tours Cedex 9, France
̧
S
* Supporting Information
ABSTRACT: In an effort toward the visualization of β-amyloid plaques by in vivo imaging techniques, we have conjugated an
optimized derivative of the Pittsburgh compound B (PiB), a well-established marker of Aβ plaques, to DO3A-monoamide that is
capable of forming stable, noncharged complexes with different trivalent metal ions including Gd3+ for MRI and 111In3+ for
SPECT applications. Proton relaxivity measurements evidenced binding of Gd(DO3A-PiB) to the amyloid peptide Aβ1−40 and to
human serum albumin, resulting in a two- and four-fold relaxivity increase, respectively. Ex vivo immunohistochemical studies
showed that the DO3A-PiB complexes selectively target Aβ plaques on Alzheimer’s disease human brain tissue. Ex vivo
biodistribution data obtained for the 111In-analogue pointed to a moderate blood−brain barrier (BBB) penetration in adult male
Swiss mice (without amyloid deposits) with 0.36% ID/g in the cortex at 2 min postinjection.
KEYWORDS: imaging agents, Gd3+, 111In3+, amyloid plaques, Alzheimer’s disease, Pittsburgh compound B
pass the blood−brain barrier (BBB). They inspired the
lzheimer’s disease (AD) is a chronic neurodegenerative
Adisorder leading to progressive decline of cognitive development of amyloid-labeling nuclear imaging probes.7−11
11C- and 18F-labeled derivatives of stilbene9 and the Pittsburgh
compound-B (PiB)7,8 are promising PET tracers of AD; certain
compounds are making their way into the clinics.8−10 Attempts
have been also made to develop 99mTc-labeled Aβ SPECT
probes, which, given the easier accessibility of 99mTc as
compared to 18F, could provide a more convenient approach
to the detection of AD. Small, neutral 99mTc-probes have been
reported based on derivatives of biphenyl,12 benzothiazole
functions.1−3 Characteristic neuropathological findings associ-
ated with AD include the formation of extracellular senile
plaques containing β-amyloid peptides and neurofibrillary
tangles.4−6 Currently, no clinical imaging techniques are
available for early detection of AD. Thus, the development of
diagnostic agents for early in vivo visualization of the amyloid
plaques is a critical issue, which would also be important for
monitoring new therapies. Significant advances have been made
in the field of nuclear imaging, in particular in positron
emission tomography (PET). Small organic compounds such as
derivatives of thioflavin T, benzoxazoles, and stilbenes have
high binding affinities for the Aβ aggregates and some of them
Received: January 31, 2013
Accepted: April 14, 2013
Published: April 15, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
aniline,13 chalcone,14 flavone,15 or pyridyl benzofurane.16 Some
showed reasonable brain uptake and affinity toward β-amyloid
plaques.16
a
Scheme 1. Synthesis of Ln(DO3A-PiB)
Magnetic resonance imaging offers noninvasive mapping of
structure and function with excellent spatial resolution. MR
contrast can be enhanced with paramagnetic contrast agents
(CAs), ideally with specific and selective delivery. Gd3+
complexes are the most widely used contrast materials.17 In
the absence of any CA, the MR contrast in the amyloid plaques
is associated to iron accumulation, which leads to hypointense
spots in T2, T2* or susceptibility-weighted images.18 The
detection of plaques weakly loaded with iron is more
challenging, and exogenous contrast agents become necessary.
So far, the development of MRI contrast agents for the
diagnosis of Alzheimer’s disease has been modest. Poduslo et al.
visualized AD plaques with GdDTPA conjugated to a
putrescine-modified human Aβ1−42 peptide able to cross the
BBB and target amyloid deposits in the brain of AD transgenic
mice. Given the large size of the probe, several days (weeks)
were necessary to label the amyloid plaques in the transgenic
mouse brain in vivo.19 Yang et al. detected amyloid deposition
with iron oxide nanoparticles coupled to Aβ1−42 peptide after
intracarotid mannitol treatment to enhance BBB permeability
in transgenic mice.20
a
Reagents and conditions: (a) THF, NEt3, 4-nitrobenzoyl chloride,
0°C, 96%; (b) chlorobenzene, Lawesson’s reagent, reflux, 85%; (c)
10% NaOH, K3Fe(CN)6, reflux, 52%; (d) SnCl2, EtOH, reflux, 92%;
(e) ClCH2COCl, K2CO3, CH3CN, r.t., 86%; (f) tBu3DO3A, K2CO3,
CH3CN, r.t., 88%; (g) dichloromethane, TFA, 45°C, 79%; (h) GdCl3,
pH 7.
Multimodality imaging combines two or more method-
ologies to provide complementary information. Optimally, the
imaging probes used in the different modalities should possess
identical biodistribution enabling an overlap of the acquired
images. Metal ions provide suitable probes for various imaging
modalities. By the appropriate choice of a chelating agent, one
can complex, with the same ligand, different metal ions that
possess properties for these imaging modalities. Provided their
charge is identical, these complexes are expected to have similar
biodistribution and are therefore interesting candidates for
multimodal imaging applications.
Our objective was to design, synthesize, and investigate novel
ligands with capability of (i) efficiently complexing metal ions
adapted to different imaging modalities, including Gd3+ for
MRI, 111In3+ for SPECT, or 68Ga3+ for PET; (ii) labeling Aβ
plaques via specific binding; and (iii) delivering the complexes
across the BBB. We have conjugated a PiB derivative21 to the
metal chelating unit DO3A-monoamide. PiB derivatives are
usually noncharged analogues of thioflavin T with excellent
BBB permeability. DOTA4− derivatives are among the best
chelators for many metal ions. They form highly stable and
inert complexes, important for safe in vivo use. In our ligand
design, the formation of neutral complexes with trivalent metal
ions and a short linkage between the metal binding and the
amyloid-recognition sites were essential elements to facilitate
BBB permeability.
1
■
Figure 1. H NMRD profiles of Gd(DO3A-PiB) at 0.2 mM ( ), 5
▼
○
mM ( ), 0.2 mM in the presence of 0.2 mM Aβ1−40 ( ), and 0.2 mM
in the presence of 0.2 mM HSA ( ) (0.05 M HEPES; pH 7.4, T =
△
310 K).
binding affinity of Gd(DO3A-PiB) to Aβ1−40 was evaluated by
surface plasmon resonance measurements (Figure S9, Support-
ing Information), which yielded Kd = (180 10) μM, a value
considerably higher than that for small benzothiazole
derivatives (Kd < 1 μM).22 Given their similar charge and the
fact that the binding interaction mainly involves the PiB moiety,
similar binding affinity is expected also for the Eu3+ and In3+
analogues. Gd(DO3A-PiB) binds to serum albumin as well,
causing a remarkable increase of relaxivity at intermediate fields
(Figures 1 and S1−S4, Supporting Information). Albumin
binding leads to a prolonged lifetime of the agent in the blood
pool that, given the slower elimination from the body, can be
useful for an MRI probe, though unfavorable for nuclear
imaging probes due to longer radiation exposure and higher
nonspecific signal. Strong HSA binding can be also detrimental
for the BBB permeability of the agent.23 The binding affinity of
Gd(DO3A-PiB) to HSA has been assessed by proton relaxation
enhancement measurements and yielded Kd = 110 20 μM
(Figures S1 and S2, Supporting Information).24 The NMRD
curve recorded at 5 mM Gd(DO3A-PiB) concentration shows
the relaxivity peak at intermediate magnetic fields, typical of
slowly tumbling systems thus evidencing the formation of
micellar aggregates. A critical micellar concentration, cmc = 1.90
The synthesis of Ln(DO3A-PiB) complexes is summarized in
Scheme 1 (see Supporting Information). Magnetic field
dependent nuclear magnetic relaxation dispersion (NMRD)
profiles are commonly used to characterize MRI contrast
agents.17 The NMRD curve of Gd(DO3A-PiB) recorded at 0.2
mM concentration is characteristic of a small-molecular weight
complex (Figure 1).
Upon binding of Gd(DO3A-PiB) to Aβ plaques, higher
relaxivity is expected since the complex becomes immobilized.
Indeed, in the presence of the amyloid peptide Aβ1−40 (cGd
=
cAβ1−40 = 0.2 mM), the relaxivity of Gd(DO3A-PiB) increases
considerably at magnetic fields where the effect of slower
rotation is most pronounced, evidencing an interaction. The
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ACS Medicinal Chemistry Letters
Letter
0.02 mM, was determined by relaxometry25 (Figure S3,
Supporting Information).
Table 1. Molecular Weight (MW) and Lipophilicity (log
Oct/H2O) of Phenylbenzothiazole (BTA) Derivatives
P
We tested the capability of Eu(DO3A-PiB) to bind amyloid
aggregates on postmortem human brain tissue of AD patients.
The staining with Eu(DO3A-PiB) revealed the presence of
various amyloid deposits. The distribution pattern and intensity
of the senile plaques were similar for PiB, thioflavin-S, and
Eu(DO3A-PiB) (Figures S6 and S7, Supporting Information).
Eu(DO3A-PiB) seems to have lower affinity for neurofibrillary
tangles, being thus more specific to Aβ deposits than PiB
(Figure 2). To confirm that PiB and Eu(DO3A-PiB) labeling is
thioflavin T
0.57
2.6
319
240
256
254
256
616
842
7
R1 = CH3, R2 = NH2
R1 = OCH3, R2 = NH2
R1 = CH3, R2 = NHCH3
R1 = OH, R2 = NHCH3 (PiB)
R1 = OCH3, R2 = ReOTEEDA
Gd(DO3A-PiB)
7, 21
1.9
7
2.6
7
1.23
2.52
−0.15
26
26
this work
The molecular weight of Gd(DO3A-PiB), MW = 842, is above
the optimal values,28 although peptide analogues with a MW >
1000 Da were shown to cross the BBB.29,30
In order to evaluate the penetration of our complexes across
the BBB, we have performed in vivo biodistribution experi-
ments with the 111In-analogue in adult male Swiss mice without
amyloid deposits in their brain. The radiolabeling of the ligand
has been carried out in isotonic ammonium acetate at pH 7.0,
by adding a solution of 111InCl3 (specific activity 15.38 TBq/
mg), to obtain 1:1 111In3+ complexes (10% ligand excess, 100
°C, 30 min). Thin layer chromatography indicated >96%
radiochemical purity (Figure S7, Supporting Information). The
complex remained stable in vitro with 2% degradation at 22 h
after radiolabeling. The in vivo biodistribution of 111In(DO3A-
PiB) at 2 and 30 min (Table 2) showed the highest uptake in
Figure 2. Positive staining of an Alzheimer tissue with PiB (a−c) and
Eu(DO3A-PiB) (d−f). Senile plaques (left), diffuse deposits (middle),
and neurofibrillary tangles (right) are put into evidence by the PIB and
Eu(DO3A-PiB) staining.
specific for the amyloid deposits, we performed a double
fluorescence staining experiment using the routinely employed
6F3D antibody (Figure 3). The staining of PiB and Eu(DO3A-
Table 2. Distribution of 111In(DO3A-PiB) in Swiss Mice 2
and 30 min after Tracer Injection; the Data Represent the
Mean SD Percent Injected Dose Per Gram (% ID/g, n =
4)
% ID/g
2 min
30 min
kidney
cerebellum
cortex
heart
12.53 1.07
0.50 0.07
0.36 0.03
7.36 0.18
4.90 0.12
14.59 0.23
11.42 0.77
4.23 0.42
0.21 0.02
0.11 0.01
1.51 0.21
7.65 1.59
4.23 0.45
1.44 0.14
liver
blood
lung
Figure 3. Micrographs illustrating the colocalization of PiB or
Eu(DO3A-PiB) and 6F3D-antibody labeling of amyloid deposits of
postmortem human brain tissue of an AD patient: the colocalization is
confirmed by the magenta color in the merge images; scale bar = 20
μm.
kidney suggesting urinary elimination. The radioactive signal
was also elevated in lungs 2 min after tracer injection, then
decreased at the 30 min time point. This transient high lung
uptake reflects the large blood volume of this blood-rich organ.
In the brain, the % ID/g in the cortex and cerebellum at 2 min
was 0.36 and 0.5, while at 30 min, it was 0.11 and 0.21,
respectively. The clearance measured by the ratio of % ID/g
values at 2 and 30 min is 3.3, comparable to that for PiB.21
99mTc-based β-amyloid tracers had similar or higher brain
uptake (0.1−1.4% ID/g at 2 min and 0.1−1.08% ID/g at 30
min).12−16 Given the higher molecular weight of 111In(DO3A-
PiB), the brain % ID/g values are remarkable. They are similar
to the cerebral biodistribution of some SPECT radioligands,
including PE2I31 and CLINDE,32 developed for brain imaging
of the dopaminergic system and neuroinflammation. Therefore,
the BBB crossing of 111In(DO3A-PiB) might be sufficient for
nuclear imaging amyloid deposits in rodent transgenic models
overexpressing Aβ aggregates. BBB permeability sufficient for
MRI detection with the Gd3+ analogue will be certainly difficult
PiB) is strong in the focal deposits, while the surrounding
crown is slightly less positive, probably due to its partially
fibrillar morphology. Some of the less intense Aβ positive
structures were not labeled by PiB or Eu(DO3A-PiB).
The BBB permeability of a compound is related to its (i)
lipophilicity, expressed by the water/octanol partition coef-
ficient, log Poct/water, (ii) molecular weight (MW), and (iii)
plasma pharmacokinetics.23 Low MW amphiphilic molecules
with log Poct/water ≈ 2 have optimal BBB penetration. For
Gd(DO3A-PiB), log Poct/water = −0.15 was obtained, a value
lower than that for phenylbenzothiazole derivatives (Table
1).7,21,26 It is similar to those reported for neutral In(DTPA-
bisamide) complexes bearing hydrophobic chains,27 and higher
than log Poct/water = −2.86 for the double-charged In(DTPA)2−.
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Letter
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amyloid deposits can be useful in animal studies where a BBB
opening is feasible. These agents can complete the use of
nonspecific Gd-complexes like GdDTPA applied in intra-
cerebroventricular injection protocols or can be useful in ex
vivo MRI to stain Alzheimer brain tissues.33
In conclusion, we synthesized a novel amyloid targeted
ligand, which is able to efficiently complex different metal ions
adapted to various imaging modalities, including Gd3+ for MRI
and 111In3+ for SPECT. The complexes bind the amyloid
peptide Aβ1−40. This results in elevated relaxivity of the Gd3+
analogue due to reduced rotational motion, which will lead to
an increased contrast in T1-weighted images upon binding to
senile plaques. The complexes show affinity for amyloid
deposits in postmortem human tissue. The biodistribution
experiments in normal mice proved moderate brain uptake.
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ASSOCIATED CONTENT
* Supporting Information
Synthesis, immunohistochemistry, relaxometric studies, and
experimental procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
*(C.F.G.C.G.) Department of Life Sciences, University of
Coimbra, P.O. Box 3046, 3001-401 Coimbra, Portugal. E-mail:
geraldes@ci.uc.pt. Fax: (+351) 2 39 85 36 07.
■
́
*(E.T.) E-mail: eva.jakabtoth@cnrs-orleans.fr. Fax: (+33) 2 38
63 15 17.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
this manuscript.
Funding
We acknowledge financial support of the F.C.T. Portugal
(Ph.D. grant SFRH/BD/46370/2008 to A.F.M.), the French-
Portuguese PESSOA project, and the European Action
TD1004 “Theragnostics Imaging and Therapy” and TD1007
“PET-MRI”.
Notes
(15) Ono, M.; Ikeoka, R.; Watanabe, H.; Kimura, H.; Fuchigami, T.;
Haratake, M.; Saji, H.; Nakayama, M. 99mTc/Re complexes based on
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plaques. Bioorg. Med. Chem. Lett. 2010, 20, 5743−5748.
(16) Cheng, Y.; Ono, M.; Kimura, H.; Ueda, M.; Saji, H.
Technetium-99m labeled pyridyl benzofuran derivatives as single
photon emission computed tomography imaging probes for β-amyloid
plaques in Alzheimer’s brains. J. Med. Chem. 2012, 55, 2279−2286.
(17) Toth, E.; Merbach, A., Eds. The Chemistry of Contrast Agents in
Medical MRI; John Wiley & Sons: Chichester, U.K., 2001.
(18) Jack, C. R.; Garwood, M.; Wengenack, T. M.; Borowski, B.;
Curran, G. L.; Lin, J.; Adriany, G.; Grohn, I. H. J.; Grimm, R.; Poduslo,
J. F. In vivo visualization of Alzheimer’s amyloid plaques by magnetic
resonance imaging in transgenic mice without a contrast agent. Magn.
Reson. Med. 2004, 52, 1263−1271.
(19) Poduslo, J. F.; Wengenack, T. M.; Curran, G. L.; Wisniewski, T.;
Sigurdsson, E. M.; Macura, S. I.; Borowski, B. J.; Jack, C. R., Jr.
Molecular targeting of Alzheimer’s amyloid plaques for contrast-
enhanced magnetic resonance imaging. Neurobiol. Dis. 2002, 11, 315−
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(20) Yang, J.; Wadghiri, Y. Z.; Hoang, D. M.; Tsui, W.; Sun, Y. J.;
Chung, E.; Li, Y. S.; Wang, A.; de Leon, M.; Wisniewski, T. Detection
of amyloid plaques targeted by USPIO-A beta 1-42 in Alzheimer’s
The authors declare no competing financial interest.
ABBREVIATIONS
■
AD, Alzheimer’s disease; CA, contrast agent; MRI, magnetic
resonance imaging; PET, positron emission tomography;
SPECT, single-photon emission computed tomography; BBB,
blood brain barrier; HSA, human serum albumin; MW,
molecular weight; PiB, Pittsburgh compound B
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