Preclinical PET as Translational Tool for Imaging Alzheimer's Disease

214 Pages·2014·28.78 MB·English

by  Andre Manook

Dissertation
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Preclinical PET as Translational Tool
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for Imaging Alzheimer’s Disease
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Fakultät für Medizin
Nuklearmedizinische Klinik und Poliklinik

Preclinical PET as Translational Tool
for Imaging Alzheimer’s Disease

Small-Animal PET Imaging of Beta-Amyloid Plaques with [11C]PiB,
its Multi-Modal Validation and
Application to the Evaluation and Ranking of New AD Tracers

André Manook

Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München
zur Erlangung des akademischen Grades eines

Doctor of Philosophy (Ph.D.)

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. med. Claus Zimmer

Prüfer der Dissertation: 1. Univ.-Prof. Dr. Markus Schwaiger
2. Univ.-Prof. Dr. Johann Förstl

Die Dissertation wurde am 14. März 2012 bei der Technischen Universität München eingereicht
und durch die Fakultät für Medizin am 23. März 2012 angenommen.
This work has been supported by Deutsche Forschungsgemeinschaft (DFG),
Karl-Max von Bauernfeind Association and Elite Graduate Network of Bavaria.
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For Carolin.
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Declaration
I declare on oath that this thesis is my own work. If not mentioned otherwise in the specific
section, every imaging procedure, experiment, pre- and post-processing and analysis of data
was performed by myself. The majority of results presented here was published in international
peer-reviewed journals by myself or in close collaboration with colleagues. Published data has
been referenced appropriately.
This work has not been submitted in any form for another degree or diploma at any university
or other institution of higher education. Information derived from published or unpublished
work of others has been acknowledged in the text and a list of references is given.

Munich, 12 March 2012 André Manook

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Abstract
Alzheimer's disease (AD) causes unprecedented ethical, social and economical implications.
The disease concept remains unclear and, hence, comprehensible therapeutic targets do not
exist. This requires new and reliable approaches to investigate the disease concept further and
to create hard evidence on postulated phenomena. Excessive production of amyloid-β (Aβ) via
proteolysis of the amyloid precursor protein (APP) and the subsequent oligomerization and
aggregation to Aβ plaques in the brain are considered to be initial pathological events and a
major early and causal hallmark of AD. Therefore, non-invasive and quantitative Aβ imaging
with positron emission tomography (PET) in humans is by now considered a helpful technique
in this quest. Preclinical Aβ imaging in rodent disease models has been limited to very few
studies and is still challenging. Recent improvements of small-animal PET (μPET) performance
have facilitated in vivo imaging of rats and mice and there is an abundance of available
transgenic (Tg) mouse models of AD. Together, they should provide for a valuable method in
translational research such as developing specific imaging markers and monitoring new
therapeutic approaches. However, intrinsic methodological constraints such as image
resolution of PET and applicability of available research models have so far limited the
feasibility of PET imaging in transgenic mouse models of AD.
The development and evaluation of a feasible protocol for small-animal PET imaging of
Aβ plaques in mouse brain is presented as a first major contribution of this thesis. For this, the
established "gold standard" radiotracer [11C]PiB was used with specific activities commonly
used in human studies. In vivo mouse brain magnetic resonance (MR) images for anatomical
reference were acquired with a clinical 1.5 Tesla (T) system. An initially unpublished,
homozygous APP/PS1 transgenic mouse line (ARTE10) with low inter-animal variability was
characterized for its potential applicability to in vivo imaging and employed to measure
Aβ burden at different disease stages in homozygous and hemizygous animals. Extensive
multi-modal cross-validations for the PET results in this mouse model with ex vivo and in vitro
methodologies, including regional brain biodistribution, dual-label digital autoradiography,
protein quantification with ELISA, fluorescence microscopy and semi-automated histological
quantification are described as a second major contribution. This includes an additional
all-in-one experimental design as a toolbox for translational applications. Employment of the
established combined methodology for the evaluation and ranking of two novel lead
compounds for AD imaging, as examples, 2-(p-[11C]Methylaminophenyl)-7-methoxy-
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Abstract
imidazo[2,1-b]benzothiazole ([11C]IBT) and 2-(4'-bromophenyl)-6-iodoimidazo[1,2-a]pyridine
([124I]BrIMPY), is shown as a third major contribution.
Specific in vivo [11C]PiB uptake in individual brain regions with Aβ plaque deposition was
demonstrated and validated in all animals of a large study cohort including homozygous AD
animals as young as nine months. Corresponding to the extent of Aβ plaque pathology, old
homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous
(23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was
shown to be suitable as an intracerebral reference region. PET results were cross-validated
and consistent with all applied ex vivo and in vitro methodologies. This also verified the validity
and applicability of the animal model for PET imaging. The reliability and validity of the
all-in-one design was verified. Specific in vivo uptake of [11C]IBT and [124I]BrIMPY in
Aβ-containing telencephalic brain regions with comparable ex vivo binding patterns to PiB and
excellent correlation with Aβ plaque pathology was shown by applying the combined
experimental concept.
The results confirm that the experimental setup for non-invasive [11C]PiB imaging of
Aβ plaques in a novel APP/PS1 mouse model of AD provides a feasible, reproducible and
robust protocol for small-animal Aβ plaque imaging. The experimental framework with this
model allows for longitudinal imaging studies with follow-up periods of approximately one and
a half years. The representative and successful evaluation of new PET and SPECT imaging
agents with high sensitivities for in vivo Aβ detection confirm that the developed experimental
design provides a toolbox for AD research in transgenic mice and, hence, the foundation for
translational Alzheimer neuroimaging.

Keywords:
Alzheimer's disease, neurodegeneration, amyloid-beta plaque, small-animal PET, tracer, PiB,
BTA, IBT, IMPY, transgenic mouse, homozygous, APP/PS1, autoradiography, biodistribution,
ELISA, histological plaque quantification
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Zusammenfassung
Die Alzheimer´sche Krankheit (Alzheimer's disease, AD) bringt beispiellose ethische,
gesellschaftliche und wirtschaftliche Auswirkungen mit sich. Das Verständnis der Erkrankung
ist weiterhin im Unklaren, weshalb nachvollziehbare therapeutische Angriffspunkte weitgehend
fehlen. Dies erfordert neue und verlässliche Ansätze zur weiteren Untersuchung verschiedener
Hypothesen und zur Untermauerung von bestehenden Postulaten der Krankheit. Die
überschüssige Produktion von Amyloid-β (Aβ) durch Abspaltung aus dem Amyloid Precursor
Protein (APP) sowie die nachfolgende Oligomerisierung und Aggregation hin zu Aβ Plaques im
Gehirn werden als anfängliche pathologische Ereignisse und als eine der frühen und
ursächlichen Kennzeichen der AD angesehen. Aus diesem Grund betrachtet man mittlerweile
die nicht-invasive und quantitative Aβ Bildgebung im Menschen, unter Verwendung der
Positronen-Emissions-Tomografie (PET), als hilfreiche Technik auf der Suche nach den
Ursachen. Allerdings ist die präklinische Aβ Bildgebung in Nagetiermodellen bisher auf sehr
wenige Studien beschränkt und weiterhin mit großen Herausforderungen verbunden. Jüngste
Verbesserungen in der Leistungsfähigkeit von Kleintier-PET Geräten (µPET) ermöglichen die in
vivo Bildgebung in Ratten und Mäusen. Im Zusammenspiel mit der Fülle verfügbarer
transgener (Tg) Mausmodelle für die AD sollte µPET eine wertvolle Methode für translatorische
Forschung sein, zum Beispiel zur Entwicklung spezifischer bildgebender Substanzen oder der
Verfolgung neuer Therapieansätze. Intrinsische methodische Beschränkungen, wie zum
Beispiel die Bildauflösung der PET und die Anwendbarkeit vorhandener Tiermodelle, haben
bisher jedoch die Brauchbarkeit der PET Bildgebung in transgenen Mausmodellen
der AD begrenzt.
Die Etablierung und Evaluation eines praktikablen Protokolls zur Kleintier-PET Bildgebung von
Aβ Plaques im Maushirn wird als der erste große Beitrag dieser Dissertation dargestellt. Hierfür
wurde der etablierte "Goldstandard" Tracer [11C]PiB mit spezifischen Aktivitäten, wie sie in
menschlichen Studien üblich sind, angewendet. Zur anatomischen Orientierung wurden in vivo
Magnetresonanz (MR) Bilder des Maushirns mit einem klinischen 1.5 Tesla (T)
Kernspintomographen aufgenommen. Eine zunächst unveröffentlichte, homozygote transgene
APP/PS1 Mauslinie (ARTE10), deren Phänotyp geringe Schwankungen zwischen den einzelnen
Tieren aufweist, wurde für ihre mögliche Anwendbarkeit in der in vivo Bildgebung
charakterisiert. Daraufhin wurden die Tiere verwendet, um den Aβ Gehalt in unterschiedlichen
Erkrankungsstadien in homozygoten und hemizygoten Tieren zu messen. Die umfangreiche
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