Neuropharmacology and Analgesia
In vitro characterisation of BF227 binding to α-synuclein/Lewy bodies

https://doi.org/10.1016/j.ejphar.2009.06.042Get rights and content

Abstract

Amyloid-β (Aβ) plaques are a pathological hallmark of Alzheimer's disease and a current target for positron emission tomography (PET) imaging agents. Whilst [11C]-PiB is currently the most widely used PET ligand in clinic, a novel family of benzoxazole compounds have shown promise as Aβ imaging agents; particularly BF227. We characterised the in vitro binding of [18F]-BF227 toward α-synuclein to address its selectivity for Aβ pathology, to establish whether [18F]-BF227 binds to α-synuclein/Lewy bodies, in addition to Aβ plaques. In vitro [18F]-BF227 saturation studies were conducted with 200 nM α-synuclein or Aβ1−42 fibrils or 100 μg of Alzheimer's disease, pure dementia with Lewy bodies or control brain homogenates. Non-specific binding was established with PiB (1 μM). In vitro binding studies indicated that [18F]-BF227 binds with high affinity to two binding sites on Aβ1−42 fibrils (KD1 = 1.31 and KD2 = 80 nM, respectively) and to one class of binding sites on α-synuclein fibrils (KD = 9.63nM). [18F]-BF227 bound to Aβ-containing Alzheimer's disease brain (KD = 25 ± 0.5 nM), but failed to bind to Aβ-free dementia with Lewy bodies or age-matched control homogenates. Moreover, BF227 labelled both Aβ plaques and Lewy bodies in immunohistochemical/fluorescence analysis of human Alzheimer's disease and Parkinson's disease brain sections, respectively. This study suggests that [18F]-BF227 is not Aβ-selective. Evaluation of BF227 as a potential biomarker for Parkinson's disease is warranted.

Introduction

Currently, there is no cure for Alzheimer's disease, an age-related neurodegenerative disease, clinically characterised by dementia. The Alzheimer's disease brain is pathologically characterised by the presence of (i) extracellular amyloid plaques comprising amyloid-β (Aβ); (ii) intracellular neurofibrillary tangles composed of hyperphosphorylated tau; (iii) synaptic loss and reactive gliosis; (iv) increased oxidative damage to lipids, proteins and nucleic acids and (v) biometal dyshomeostasis (Goedert and Spillantini, 2006).

Definitive diagnosis of Alzheimer's disease and related dementias still relies upon postmortem examination. As new therapeutic strategies undergo clinical evaluation, considerable effort is now focused on biomarkers for the early and accurate diagnosis of Alzheimer's disease, as well as therapeutic monitoring. Modern molecular imaging procedures such as positron emission tomography (PET), may provide new insight into Alzheimer's disease by non-invasively identifying the underlying pathology of these diseases in the living. Of late, Pittsburgh compound B [PiB] has proven to be a successful biomarker for the in vivo quantitation of Aβ burden (Klunk et al., 2004, Rowe et al., 2007). Nonetheless, its widespread clinical use is impracticable due to the 20-minute decay half-life of carbon-11, limiting its use to centres with an on-site cyclotron. [18F]-FDDNP also highlights Aβ deposits in the human brain; however, FDDNP also binds to neurofibrillary tangles (Agdeppa et al., 2001), as well as PrPsc (Boxer et al., 2007, Bresjanac et al., 2003). Whilst labelling with a longer half-life isotope [18F] proves to be advantageous, FDDNP's lack of selectivity considerably reduces its ability to provide differential diagnosis of neurodegenerative diseases. Hence, the development of a specific and selective [18F]-labelled imaging agent(s) for molecular Aβ imaging is highly desirable to improve diagnostic accuracy and accelerate discovery and monitoring of therapeutics.

Recently, a novel series of benzoxazole compounds have been developed as PET imaging agents; namely BF227 [2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy] benzoxazole] has been demonstrated to bind to Aβ1–42 fibrils (with low nanomolar affinity) and Aβ plaques in Alzheimer's disease brain sections (Kudo et al., 2007). [11C]-BF227-PET demonstrated retention in cerebral cortices of Alzheimer's disease patients with very little retention in normal patients; suggesting BF227 as a promising PET imaging agent for the in vivo detection of Aβ pathology in Alzheimer's disease patients. Whilst the specificity of BF227 binding to Aβ has been established, there is limited knowledge regarding its selectivity; particularly since Alzheimer's disease has been described as a ‘triple brain amyloidosis’ (Trojanowski, 2002), comprising Aβ, tau and α-synuclein that when misfolded, comprise the principal components of senile plaques, neurofibrillary tangles and Lewy bodies. Furthermore, the majority of dementia with Lewy bodies cases exhibit extensive cortical Aβ deposition along the pathognomonic Lewy bodies (McKeith et al., 2005). Hence, critical assessment of new radiotracers such as BF227 is warranted to avoid misinterpretation of results and/or incorrect diagnosis. Whilst BF227 binding to neurofibrillary tangles has previously been examined (Kudo et al., 2007), the potential of BF227 binding to α-synuclein has not been assessed. The aim of this study was to test the ability of BF227 to bind/recognise α-synuclein fibrils/Lewy bodies to establish whether [18F]-BF227 is selective for Aβ pathology.

Section snippets

Materials

All reagents were purchased from Sigma (St. Louis, MO), unless otherwise stated. Human Aβ1–42 was purchased from the W. M. Keck Laboratory (Yale University, New Haven, CT).

Characteristics of [18F]-BF227 Binding to Recombinant α-Synuclein and Aβ1–42 Fibrils

To investigate the selectivity of BF227, we tested the ability of [18F]-BF227 to bind to synthetic α-synuclein and Aβ1–42 fibrils; the major component of Lewy bodies and senile plaques, respectively. The successful formation of fibrils was determined by ThT fluorescence and transmission electron microscopy, prior to conducting the binding assays (data not shown).

Assessment of [18F]-BF227 binding was conducted using equimolar concentrations (200 nM, ∼4.0 × 10−11 mol) of either α-synuclein or Aβ

Discussion

The ongoing quest for specific and selective PET imaging agents is imperative for the early diagnosis, treatment, development and monitoring of neurodegenerative diseases, such as Alzheimer's disease. To date, the design and testing of PET imaging candidates have suggested that compounds based on histological dyes whilst specific, are not selective for Aβ pathology. Benzoxazole compounds/derivatives represent a promising new family of imaging agents that may overcome some of the limitations of

Acknowledgments

The authors would like to thank Prof. Catriona McLean, Fairlie Hinton and Geoff Pavey from the National Neural Tissue Resource Centre for sourcing and preparation of the human brain tissue. We acknowledge the funding from the National Health and Medical Research Council and Ministry of Health, Labour and Welfare, Japan. RC is an NHMRC Senior Research Fellow.

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