Characterization of a brain permeant fluorescent molecule and visualization of Aβ parenchymal plaques, using real-time multiphoton imaging in transgenic mice

Article abstract of DOI:10.1021/ol501264q

Emerging paradigms mandate discovery of imaging agents for diagnosing Alzheimer's disease (AD) prior to appearance of clinical symptoms. To accomplish this objective, a novel heterocyclic molecule (4) was synthesized and validated as Aβ targeted probe. The agent shows labeling of numerous diffuse Aβ plaques in confirmed AD human brain tissues and traverses the blood-brain barrier to enable labeling of parenchymal Aβ plaques in live mice (APP ^±/PS1^±) brains.

Full text of DOI:10.1021/ol501264q

Letter
pubs.acs.org/OrgLett
Characterization of a Brain Permeant Fluorescent Molecule and
Visualization of Aβ Parenchymal Plaques, Using Real-Time
Multiphoton Imaging in Transgenic Mice
G.S.M. Sundaram,†,‡ Kanchan Garai,§ Nigam P. Rath,○ Ping Yan,∥ John R. Cirrito,∥,#,∇
Nigel J. Cairns,∥,⊥,# Jin-Moo Lee,‡,§,∥ and Vijay Sharma*,†,‡,§
‡
§
†BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology, Department of Biomedical Engineering,
∥Departments of Neurology and Pathology & Immunology, Knight Alzheimer’s Disease Research Center, and Hope Center for
⊥
#
∇
Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110, United States
○Departments of Chemistry & Biochemistry, University of Missouri, St. Louis, Missouri 63121, United States
S
* Supporting Information
ABSTRACT: Emerging paradigms mandate discovery of imaging
agents for diagnosing Alzheimer’s disease (AD) prior to appearance
of clinical symptoms. To accomplish this objective, a novel
heterocyclic molecule (4) was synthesized and validated as Aβ
targeted probe. The agent shows labeling of numerous diffuse Aβ
plaques in confirmed AD human brain tissues and traverses the
blood−brain barrier to enable labeling of parenchymal Aβ plaques in
live mice (APP /PS1 ) brains.
of disease modifying treatments, a noninvasive PET imaging
lzheimer’s disease (AD) is the most frequent form of
Adementia, which affects 24 million people worldwide and technique to quantify brain amyloid deposition is desired.
also lacks effective therapeutic interventions.1 The failure of
clinical drug trials to reverse clinical symptoms indicates that for a
given treatment to be effective, it most likely needs to be
prescribed at a preclinical stage before the symptomatic
expression of the disease. Therefore, there is an urgent need to
identify and validate biomarkers present at preclinical stages of
this disease. AD is characterized neuropathologically by β-
amyloid (Aβ) plaques and intraneuronal accumulations of
abnormal neurofibrillary tangles (NFTs), composed of the tau
protein.2 In vitro and rodent models indicate that the
accumulation of Aβ1−42 and Aβ1−40 fragments may lead to
neuronal and synaptic loss, resulting in cognitive decline.
Additionally, model systems indicate that Aβ aggregation is an
initiating event in the AD pathogenic cascade: (a) overexpression
of amyloid precursor protein (APP; a transmembrane protein
encoded on chromosome 21) is a characteristic of Down
Syndrome (DS);3 (b) missense mutations in APP also induce an
elevation of Aβ levels;4,5 (c) mutations in the presenilin (PSEN1
Toward this objective, radiopharmaceuticals such as, [11C]2-
(4′-methylaminophenyl)-6-hydroxybenzothiazole, ([11C]PIB),9
2-(1-(6-((2[18F]fluoroethyl)methylamino)-2-naphthyl)-
ethylidene)malononitrile ([18F]FDDNP),10 [11C]4-N-methyla-
mino-4′-hydroxystilbene (SB-13),11 and (E)-4-(2-(6-(2-(2-(2-
[18F]-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-meth-
ylbenzenamine ([18F]Avid45),12 and 2-(2-fluoro-6-
(methylamino)pyridine-3-yl)benzofuran-6-ol(1 8 F-
AZD4694)13,14 have been investigated in humans using PET
imaging. In addition, [125I/131I]TZDM and [125I]IMPY have also
been investigated for SPECT applications.10 While [11C]PIB has
been most intensely studied, [18F]Avid45 and 18F-flutemeta-
mol15,16 were recently approved by FDA for Aβ imaging.
Importantly, both [18F]Avid4517 and [11C]PIB show promising
results in humans and excellent correlation with FDG.9 While
showing merit, each of these agents has demonstrated short-
comings worthy of consideration. Among these limitations,
[11C]PIB,18 [11C]SB-13,11 and [18F]Avid4512 have demonstra-
ted low biological half-lives in serum. While metabolites of PIB
have been postulated to not penetrate the brain,18 two
metabolites of [18F]Avid45 (desmethylated, 4.5%ID/g; acety-
lated analogue, 3.3%ID/g at 2 min in normal mice)12 have been
shown to permeate the brain, which could complicate the
analysis. Additionally, systematic investigations of [3H]PIB (at
tracer concentrations comparable with PET imaging) binding to
human neuropathological brain specimens indicate the inter-
and PSEN2) genes have been shown to increase the expression of
5,6
variant Aβ1−42
;
and (d) transgenic mice overexpressing APP
develop AD-like neuropathology.7 Overall, there is ample
evidence that suggests AD pathological changes (Aβ deposition
and NFT formation) occur years prior to onset of symptoms.8
While all these observations suggest that Aβ plaque formation
occurs decades prior to the beginning of neurodegeneration
phase, the temporal relationship between amyloid deposition,
neuronal loss, and cognitive decline are intensely debated and
continuously investigated. Therefore, for elucidation of these
relationships, early diagnosis of AD and the efficient assessment
Received: May 5, 2014
Published: July 8, 2014
© 2014 American Chemical Society
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Letter
action of a radiotracer with classical Aβ plaques, NFTs (intensity
is low compared with Aβ pathology), and cerebrovascular
amyloid angiopathy (CAA).19 The other Aβ imaging agent,
[18F]FDDNP, shows binding to NFTs, prion plaques, and
fibrillar Aβ, therefore indicating a lack of specificity toward
probing AD.10,20 Significantly, all agents demonstrate consid-
erable white matter binding ([18F]Avid45 shows 2-fold higher
binding than [11C]PIB), thus providing challenges for analysis in
early stages of AD. Noticeably, [11C]PIB has been unable to
detect cerebral Aβ in some patients.21 Overall, existing agents
have not demonstrated sensitivity to detect the earliest Aβ
pathophysiology to enable benefits at preclinical stages. There-
fore, F-18-incorporated ultrasensitive PET probes capable of
targeting highly prevalent sites on Aβ yet displaying faster
kinetics from nontargeted regions (high signal-to-noise ratio),
minimal white matter interaction, and targeting diffuse- as well as
Aβ fibrillar plaques could provide a quantitative amyloid imaging
test for guiding management of AD. Herein, we report chemical
characterization and crystal structure of the molecule 4 ((E)-5-
(2-(6-(2-fluoroethoxy)benzo[d]thiazol-2-yl)vinyl)-N,N-dime-
thylpyridin-2-amine), its preliminary assessment of target
specificity in mice and human tissues. Within minutes post-
intravenous injection, 4 penetrates the BBB to label Aβ plaques
in brains of APP /PS1 mice.
Figure 1. Projection view of 4 showing the crystallographic numbering
scheme of atoms (thermal ellipsoids: 50% probability).
literature procedure23) in the presence of cesium carbonate to
obtain 4. All intermediates, 1, 2, 3, and the final compound 4,
were characterized via standard analytical methods. Additionally,
4 was also analyzed for uniformity and purity, on a semi-
preparative C-18 column (Vydac), using an HPLC system.
Compound 4 eluted as a single chemical entity, with a retention
time of 10 min, thus indicating purity of the molecule. The
organic molecule 4 crystallized in a triclinic space group P1. The
ORTEP drawing showing the crystallographic numbering
scheme for 4 is illustrated in Figure 1. The crystal structure
indicates a nearly planar molecule with a calculated root-mean-
square deviation of 0.062 Å for the entire core (all atoms except
the H and F atoms) and also indicates a weak π−π interaction
and hydrogen bonding (Supporting Information, Figure 2). The
NMR spectral data of 4 were also consistent with the crystal
structure, thus indicating the presence of identical structures
both in solid and solution state.
Our strategic design for generation of an Aβ-targeted agent
involved five functional components: (a) a benzothiazole moiety
without the methyl group on the heterocyclic nitrogen of
thioflavin T to remove the positive charge for enhancing affinity
of the probe to Aβ plaques and hydrophobicity to facilitate BBB
penetration; (b) modifications of substituents on the sixth
position of the benzothiazole ring insignificantly impact affinity
of probes for plaques; (c) introduction of an olefin bond between
the benzothiazole moiety and an aromatic ring to increase
electron density as well as flexibility of the molecule to promote
interactions with binding sites present on both diffuse and
fibrillar Aβ plaques; (d) substituting a basic dimethylamino
group into an aromatic ring at the para position to the olefinic
carbon to increase electron density on the nitrogen; and (e)
incorporation of a heteroatom, such as nitrogen atom in the
aromatic ring ortho to the basic dimethylamino group, to enable
better resonance stabilization for influencing π−π interactions
for targeting of high density and moderate affinity sites on Aβ
plaques. When the above-mentioned characteristic features were
incorporated into a template scaffold, compound 4 was
synthesized as shown in Scheme 1 (synthetic chemistry details
in Supporting Information).
For assessing the ability of 4 to bind Aβ plaques, preliminary
binding assays with preformed Aβ1−42 aggregates were
performed. Following excitation at 410 nm, the fluorescence
spectrum of 4 recorded in PBS showed an emission peak with
E
max at 503 nm. Upon incubation with preformed aggregates of
Aβ1−42, the peak at 503 nm shifted slightly (Emax 485 nm) and
simultaneously demonstrated a remarkable enhancement in the
fluorescence, due to binding to Aβ aggregates (Supporting
Information, Figure 3). This enhancement in fluorescence upon
interaction with Aβ1−42 fibrils is consistent with profiles observed
with other fluorescent molecules under similar conditions.24
Additionally, no fluorescence was observed using Aβ aggregates
alone in PBS following excitation at 410 nm (negative control).
Overall, binding assay of 4 with preformed Aβ1−42 aggregates
indicates a nearly saturable binding with Kd = 59
7 nM
(Supporting Information, Figure 4).
The double-transgenic mice having coexpression of the
mutated genes (APP /PS1 ) exhibit a strikingly accelerated
accumulation of Aβ deposits compared with the single APP
transgenic counterparts.25−,27 Noticeably, several Aβ ligands and
disease-modifying therapeutics have been investigated for their
efficacy using APP /PS1 mice.28,29 Further, we assessed the
ability of 4 to stain ex vivo brain sections (50 μm) of age-matched
APP /PS1 mice and their WT (BL/6) counterparts, using
established procedures.30 As a positive control, anti-Aβ
monoclonal antibody (mHJ3.4 conjugated to Alexa 568) was
used.31 Brain sections of 24-month-old APP /PS1 mice showed
abundant staining of Aβ (Figure 2) compared with none in WT
counterparts (data not shown). Similarly, 4 (1 μM) demon-
strated abundant staining of fibrillar plaques in the cortical
regions of brain sections (Figure 2) in APP /PS1 mice. By
comparison, 4 showed no staining in WT mice (Supporting
Information, Figure 5), thus indicating its target specificity.
Finally, 4 also showed significant correlation (Figure 2; merged
image) with anti-Aβ antibody. However, a slightly less than 1:1
correlation in the merged image could be attributed to binding
patterns of 4, a small organic molecule versus an anti-Aβ
antibody.
For synthesis, 6-methoxy-2-methylbenzothiazole (1) was
obtained using literature procedures22 and condensed with 6-
(dimethylamino)nicotinaldehyde in an aqueous potassium
hydroxide (50%) solution dissolved in DMSO to obtain 2.
Following purification, 2 was demethylated in the presence of
BBr3 to yield the phenolic derivative 3. Finally, 3 was alkylated
with 2-fluoroethyl-4-methylbenzenesulfonate (prepared using
Scheme 1. Chemical Synthesis of Heterocyclic Molecule 4
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Organic Letters
Letter
PET imaging of mildly demented individuals prior to onset of
symptoms.8
The discovery and development of new ligands to enable
imaging of biomarkers in brain mandates interrogation of their
ability to permeate the BBB, excretion from nearby nontargeted
regions of the brain to provide a high signal/noise ratio, and
specificity for a given target. For assessing viability of molecules
as imaging probes in vivo,37 various imaging modalities, such as
nuclear imaging (PET/SPECT), optical imaging, and MRI have
been used to investigate simultaneously distribution kinetics and
target-receptor specificity. While the resolution of PET and MRI
allow in vivo imaging at relatively lower resolution, the
multiphoton microscopy enables precise evaluation of the
specificity of a given ligand binding to its target at a
submicrometer resolution.38 Therefore, this technique enables
characterization of probes in small animal models at a
significantly high spatial and temporal resolution.38
Figure 2. Staining of brain tissue sections (50 μm) from APP /PS1
mice using 4 (1 μM) or immunostained with mouse anti-Aβ monoclonal
antibody (mHJ3.4) conjugated to Alexa Fluor 568 (positive control).
Arrows indicate labeling of Aβ plaques. Similar results were obtained in
more than five independent experiments.
For assessing the ability of 4 to penetrate the BBB, label Aβ
parenchymal plaques, and simultaneously interrogate the
pharmacokinetic profiles from nearby brain regions, direct real-
time imaging was performed in transgenic APP /PS1 mice.
Prior to imaging, dextran−Texas Red conjugate (33 mg/kg;
dissolved in PBS to mark the blood vessels) and 4 (2 mg/kg;
dissolved in 20% DMSO in propylene glycol39) were intra-
venously administered to anesthetized APP /PS1 mice (with
cranial windows; Supporting Information). Compared with
barely detectable autofluorescence levels in either vehicle treated
BL6 (WT) mice (data not shown) or prior to imaging transgenic
APP /PS1 mice (Figure 4), bright fluorescence appeared in
large and small blood vessels of the brain following injection.
Within minutes, the fluorescent molecule crossed the BBB and
entered the brain parenchyma and Aβ deposits were labeled.
While the complete labeling of CAA occurred instantaneously
following injection, the labeling of parenchymal plaques peaked
at approximately 10 min. Overall, these data demonstrate in real
time that 4 enters the CNS rapidly and labels parenchymal Aβ
plaques. These real-time pharmacokinetic data in transgenic
mice provide an ideal platform for PET tracer development.
In summary, novel heterocyclic fluorescent molecule 4
demonstrates binding to both diffuse and fibrillar Aβ plaques, a
promising feature that could enable diagnosis of AD at preclinical
stages. Multiphoton imaging shows real-time pharmacokinetics
of 4 in brains of transgenic mice at a highest resolution,
penetration of the BBB, and ability of the agent to label Aβ
plaques in brain parenchyma and blood vessels (CAA).
Additionally, the low level of background fluorescence from
residual retention of 4 suggests high signal-to-background ratios,
an asset for PET imaging applications. Although imaging of 4
with multiphoton microscopy does not predict the sensitivity
levels obtainable with tracer imaging, the technique allows
detection of individual plaques within a very small volume of the
brain compared with PET imaging probes that provide an
averaged intensity of Aβ binding at a low spatial resolution. Thus,
Figure 3. Binding of anti-Aβ antibody (10D5, Eli Lilly, A), 4 (B), and
thioflavin S (C) to Aβ plaques in AD brain near/adjacent tissue sections.
Amyloid in post-mortem frontal lobe of a 90-year-old male.
Magnification: 200×. (A) Diffuse Aβ plaques and an arteriole with Aβ
deposits (cerebral amyloid angiopathy); 10D5 immunohistochemistry.
(B) Section stained with 4 containing the same arteriole as in (A). There
are numerous diffuse Aβ plaques and the vessel is also stained. (C)
Thioflavin S reveals amyloid in blood vessels but diffuse plaques are only
weakly stained. The same diffuse Aβ plaque is labeled in A−C. Similar
results were obtained in more than five independent cases.
Despite showing promising affinity in vitro, initial good
penetration as well as clearance in normal mice, and staining to
transgenic mice brain cross-sections, few Aβ-targeted 99mTc-
ligands have not displayed any binding with Aβ plaques in human
tissues, thus impeding their further development.32 To further
assess the ability of 4 to label Aβ plaques in human brain, staining
experiments were performed with post-mortem tissues from
clinically characterized AD patients.33,34 As a positive control, a
highly specific anti-Aβ antibody (10 D5, Eli Lilly, Indianapolis,
IN) was used to ascertain the presence of Aβ plaques (Figure
3A), using well-established procedures.35 Importantly, 4 (10
μM) demonstrated strikingly distinct labeling of Aβ plaques in
the tissue sections of frontal lobe of a 90-year-old male with AD
(Figure 3) and absence of Aβ plaques in normal controls
(Supporting Information, Figure 6), thus indicating target
specificity. Noticeably, 4 also indicated proficient and distinct
labeling of plaque and leptomeningeal vessels (cerebral amyloid
angiopathy; CAA) (Figure 3B). These data are consistent with
other unlabeled counterparts of FDA-approved PET agents.36
Furthermore, thioflavin S, an amyloid staining dye, showed
staining of amyloid in the blood vessel (CAA) and indicated only
weak staining of diffuse plaques (Figure 3C). Importantly, 4
demonstrated labeling of numerous diffuse Aβ plaques (Figure
3B). Overall, the ability and sensitivity of 4 to detect diffuse
plaques could represent an important advancement to enable
Figure 4. Real time imaging of 4 in brains of APP /PS1 transgenic mice: Following labeling of blood vessels with dextran-Texas Red, 4 (2 mg/kg) was
intravenously injected. A z-stack image series was acquired using an LSM 510META NLO microscope (Carl-Zeiss Inc.).
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Letter
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental and synthetic procedures (1−4), analytical data,
and X-ray crystallographic data for 4. This material is available
free of charge via the Internet at http://pubs.acs.org.
(19) Lockhart, A.; Lamb, J. R.; Osredkar, T.; Sue, L. I.; Joyce, J. N.; Ye,
L.; Libri, V.; Leppert, D.; Beach, T. G. Brain 2007, 130, 2607−15.
(20) Agdeppa, E.; Kepe, V.; Petric, A.; Satyamurthy, N.; Liu, J.; Huang,
S.; Small, G.; Cole, G.; Barrio, J. Neuroscience 2003, 117, 723−730.
(21) Cairns, N. J.; Ikonomovic, M. D.; Benzinger, T.; Storandt, M.;
Fagan, A. M.; Shah, A. R.; Reinwald, L. T.; Carter, D.; Felton, A.;
Holtzman, D. M.; Mintun, M. A.; Klunk, W. E.; Morris, J. C. Arch.
Neurol. 2009, 66, 1557−62.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 314-362-9358. E-mail: sharmav@mir.wustl.edu.
Notes
The authors declare no competing financial interest.
(22) Ma, D.; Xie, S.; Xue, P.; Zhang, X.; Dong, J.; Jiang, Y. Angew.
Chem., Int. Ed. 2009, 48, 4222−5.
ACKNOWLEDGMENTS
■
Financial assistance for this work was provided by grants from the
National Institutes of Health in part by AG030498 (V.S.) and
AG033328 (V.S.), P50-AG05681, P01-AG03991 (N.J.C.), and
MIR funds (V.S.).
(23) Yu, K.; Park, J.; Yang, S. Bull. Korean Chem. Soc. 2004, 25, 506−
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