- Email: [email protected]
PET imaging of amyloid in Alzheimer's disease
Reviews PET imaging of amyloid in Alzheimer’s disease
Agneta Nordberg Alzheimer’s disease (AD) is the most common form of dementia and is characterised by progressive impairment in cognitive function and behaviour. The pathological features of AD include neuritic plaques composed of amyloid- peptide (A) fibrils, neurofibrillary tangles of hyperphosphorylated tau, and neurotransmitter deficits. Increases in the concentration of A in the course of the disease with subtle effects on synaptic efficacy will lead to gradual increase in the load of amyloid plaques and progression in cognitive impairment. Direct imaging of amyloid load in patients with AD in vivo would be very useful for the early diagnosis of AD and the development and assessment of new treatment strategies. Three different strategies are being used to develop compounds suitable for in vivo imaging of amyloid deposits in human brains. Monoclonal antibodies against A and peptide fragments have had limited uptake by the brain when tested in patients with AD. When putrescine-gadolinium-A has been injected into transgenic mice overexpressing amyloid, labelling has been observed with MRI. The small molecular approach for amyloid imaging has so far been most successful. The binding of different derivatives of Congo red and thioflavin has been studied in human autopsy brain tissue and in transgenic mice. Two compounds, fluorine-18-labelledFDDNP and carbon-11-labelled-PIB, both show more binding in the brains of patients with AD than in those of healthy people. Additional compounds will probably be developed that are suitable not only for PET but also for single photon emission CT (SPECT). Lancet Neurol 2004; 3: 519–27
Alzheimer’s disease (AD) accounts for 60–70% of all dementia and is characterised by irreversible memory impairment, continuous cognitive decline, and behavioural disturbances. The duration of the disease can vary between 5 years and 20 years. About 6% of the population of age over 65 years have dementia and the number of new cases in 2000 was estimated to be 4·6 million worldwide.1 Because of the increase in the number of elderly people, it is estimated that the worldwide number of patients with dementia (25 million in 2000) will increase to 63 million in 2030 and to 114 million in 2050.1 The increasing number of people with dementia is a great challenge for society and health-care systems. Although there has been a rapid increase in the understanding of the aetiology, genetics, and underlying pathophysiological mechanism for AD during recent years there is still no cure for the disease. Therapy is mainly symptomatic and great efforts are made to develop new
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drugs that lower amyloid load in the brain and that have neuroprotective properties to slow disease progression. Early detection of AD will be a prerequisite for early treatment. Several compounds have been developed for the imaging of amyloid: radiolabelled amyloid- peptide (A) antibodies and peptide fragments; small molecules (such as derivatives of Congo red, thioflavin, stilbene, and acridine) for PET and SPECT imaging; and compounds for MRI. The aim of this review is to describe the search for and development of amyloid imaging compounds, which, after studies in post-mortem human brain tissue and transgenic APP mice, finally led to PET studies in patients. Although there are limitations to amyloid-imaging technology, and further validation and development is needed for routine clinical assessment, imaging should be used to assess the effectiveness of antiamyloid therapy.
The amyloid hypothesis The presence of amyloid in a form of dementia (which later became known as AD) was described by Alois Alzheimer.2 The German physician, described the presence of amyloid plaques and neurofibrillary tangles in the brain of a 51-yearold woman named Auguste D who had a history of progressive memory impairment.3 The extracellular plaques and deposits and intracellular neurofibrillary tangles became the hallmark pathological features of AD together with neuronal and synaptic losses and neurotransmitter deficits.4,5 Purified and sequenced by Glenner and Wong in 1984,6 amyloid is present in plaques as small insoluble A peptides.7 Amyloid deposits can be morphologically classified into neuritic plaques, amyloid angiopathy of the capillaries, amyloid angiopathy of the arteries and veins, and diffuse amyloid deposits.8 The depositing of A may be an early and obligatory event in the pathogenesis of AD.9 A gradual cortical depositing of amyloid was described by Braak and Braak.9 They proposed that amyloid deposits first appeared in the basal neocortex, and spread to all areas of the cortex (figure 1);9 at later stages the deposits also involved subcortical brain regions, including the
AN is at the Karolinska institute, Neurotec Department, Division of Molecular Neuropharmacology, Karolinska University Hospital Huddinge, Stockholm, Sweden. Correspondence: Prof Agneta Nordberg, Karolinska institute, Neurotec Department, Division of Molecular Neuropharmacology and Department of Geriatric Medicine, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. Tel +46 8 58585467; fax +46 8 6899210; email [email protected]
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Review
PET imaging of amyloid
impairment of brain glucose metabolism at presymptomatic stages years before clinical symptoms.23–25 My colleagues and I have recently observed impairment in cerebral glucose metabolism in presymptomatic carriers of a PS1 mutation, 10–15 years before estimated onset of AD (unpublished). Progressive impairment of cerebral glucose metabolism in people with the Swedish mutation can be visualised with PET (figure 4). People who have the (APOE) ⑀4 allele have low rates of cerebral glucose metabolism in their brains decades before a possible onset of AD.26–28 Early abnormal patterns of glucose metabolism in people at risk of AD highlight the importance of identifying AD at a Figure 1. Evolution of amyloid deposits in AD. From the analysis of the presence of amyloid in presymptomatic stage. brains obtained at autopsy three stages can be distinguished in the gradual development of cortical amyloid deposition in AD. Courtesy of Dr Heikki Braak, Department of Pathology, People with mild cognitive Frankfurt University, Frankfurt, Germany. impairment (MCI) have a high risk of AD.29 MCI is an intermediate stage cerebellum.10 The distribution of plaques differs from that of between cognitive change with normal ageing and what neurofibrillary tangles.11 The finding of genetic mutations might constitute an early stage of AD. The distinction that cause AD by affecting the processing of amyloid between normal ageing and MCI might be quite subtle. precursor protein (APP) led to the amyloid cascade Compared with control individuals, people with MCI have hypothesis12 with increased production of A1–42, A1–40, impairments in memory function and are more likely to amyloid fibrils, and amyloid plaques (figure 2). Amyloid carry the APOE ⑀4 allele.30 Morphological and glucose pathology is assumed to precede the formation of tau- metabolism changes are found in patients with MCI who positive paired helical filaments (neurofibrillary tangles), will develop AD.31–33 These people also have amyloid pathogenetic microglial processes, and oxidative stress pathology in higher amounts than expected for age at reactions.13 The APP 670/671 (Swedish) mutation14 autopsy but have preserved cholinergic enzymes.34 In order produced highly aggregable forms of A1–42 and more and to understand and detect early AD, not only functional larger plaques than in sporadic AD15–17 (figure 3). A crucial consequences of the pathological processes but also in vivo question is whether soluble or insoluble oligomers or mature neurochemistry and imaging of aetiological and pathological amyloids are most neurotoxic (figure 2). Measurement of processes including A pathology need to be measured.34–39 A concentrations in post-mortem brain tissue revealed The possibility of imaging and quantifying amyloid in high concentrations of A1–42 and A1–40 in early stage of living patients with AD will enable a critical assessment of dementia associated with cognitive decline.18,19 Subtle the amyloid hypothesis. Amyloid imaging as a surrogate changes in soluble oligomers of A might lead to synaptic marker will provide presymptomatic detection and allow dysfunction in the brains of patients with AD.20 The amount studies of disease progression. Amyloid imaging in people of amyloid deposits may not be of relevance for status of from families with hereditary forms of AD including cognition although a strong association has been reported chromosomal aberrations (APP and PS1 mutations) between amyloid load in entorhinal cortex and cognition.21 as well as carriers of APOE ⑀4 is therefore of highest Studies to assess the benefits of antiamyloid therapy, the priority. Ongoing studies with antiamyloid therapy, such as visualisation of amyloid load, and its association with vaccination, have shown some difficulties in the assessment cognitive effects are needed. of drug effects at autopsy. The visualisation of amyloid plaques in the brains of living patients with AD would Early detection of AD greatly aid the assessment of efficacy for antiamyloid The rapid recent development of non-invasive tools for the therapy. imaging of human brains has had a great effect on our ability to investigate and understand brain function. Structural Amyloid imaging brain imaging, such as MRI as well as functional brain An ideal imaging ligand should fulfil several criteria (panel). imaging with single photon emission CT (SPECT) and PET In the past 10 years several different strategies have been have revealed and increased the understanding of early used to develop compounds suitable for amyloid imaging changes in AD. Longitudinal studies in families with AD with different chemical structures and properties (figure 5). caused by mutations in the APP and presenilin genes The binding properties of the compounds have been studied have shown evidence for structural changes22 and in vitro by autoradiographic ligand studies with tissue
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Review
PET imaging of amyloid
APP N APPs
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C  secretase
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Figure 3. Neuritic plaques and astrocytes visualised by immunostaining with antibodies against A and GFAP in thin tissue paraffin fixed autopsy AD brain tissue. More and larger neuritic plaques were observed in the temporal cerebral cortex of a 62-year-old woman with the Swedish mutation14 than in a 66-year-old man with sporadic AD (inserted figure). More astrocytes were distributed around the amyloid plaques in the brain of the patient with genetic AD.
Protofibrils
Amyloid fibrils
Amyloid plaques Intracellular pool of A [A] soluble
Extracellular pool of A [A] soluble [A] insoluble
Figure 2. APP processing pathways and formation of A peptide. APP is initially cleaved by ␣ and  secretases, generating the soluble fragments ␣APP and APPs and the C-terminal fragments C83 and C99. Further ␥−secretase processing of the C-terminal fragments releases p3 and A respectively. A protofibrils have a secondary structure rich in -sheets that can give rise to the amyloid fibrils present in plaques.
homogenates or thin slices from mice or human brains. Labelled compounds have been injected into mice to study in vivo uptake and binding. Some promising compounds have been visualised with different imaging techniques in mice, monkeys, and patients with AD (table).39–47 The development of plaque-binding compounds started with monoclonal antibodies against A and peptide fragments,48–50 which were followed by small radiolabelled analogues of Congo red, chrysamine-G, and thioflavin42,51 applicable for SPECT and PET, and molecules targeting amyloid plaques suitable for MRI.43,44 The compounds fluorine-18-labelled FDDNP41 and the Pittsburgh Compound-B (carbon-11-labelled PIB)39 have both been given to human beings and the uptake and binding in brain have been studied by PET in patients with AD (table).
temporal cortex was observed.48 Antibodies to A1–28 labelled with technetium-99 were developed by Friedland and co-workers.49 The monoclonal antibody 10H3 was identified as a promising amyloid imaging agent.40 When the compound was given to six people with probable AD, SPECT studies revealed an uptake of 99Tc-10H3 solely around the scalp and cranial bone marrow, whereas no cerebral uptake was observed.40 In order to increase the brain permeability to A1–40, Wengenack and co-workers52 used putrescine modified A1–40, which showed higher permeability to brain than the natural peptide and labelled amyloid deposits in transgenic APP/PS mice.52 Similarly, when a conjugation of iodine-125-labelled A1–40 to 8D3 monoclonal antibody was used, uptake of the peptide and binding to A was observed in the brain of transgenic mice.50 Lovat and co-workers53 imaged amyloid in peripheral tissue of patients with systemic amyloidosis by use of iodine-123-labelled serum amyloid P component (SAP) and SPECT. The same ligand was used for imaging of amyloid in the brains of patients with AD. No binding of 123I-SAP was observed,54 probably because of poor passage across the blood–brain barrier.55 However, Bornebroek and co-workers56 reported accumulation of 123I-SAP in the cerebral cortex when it was given to two patients with hereditary cerebral amyloid angiopathy (Dutch type), which was imaged with SPECT. Labelling of cerebral amyloid has been observed in transgenic APP mice after intranasal injection of 125I-basic fibroblast growth factor (125I-bFGF) and SAP.54 The authors suggest that intranasal injection of radiolabelled compounds might be promising for the imaging of brain amyloid in human beings.57 Detection of brain amyloid by MRI
Radiolabelled A antibodies and peptide fragments
Antibodies to A have been tested as suitable imaging agents for amyloid. A saturable binding of iodine-125-labelled A1–40 protein to tissue homogenates prepared from AD
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Magnetic resonance microscopy (MRM) produces images with higher spatial resolution than conventional MRI. When MRM was applied in human brain autopsy tissue from five patients with AD and three age-matched control individuals
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Review
PET imaging of amyloid
Criteria for an ideal imaging compound for the detection of amyloid in the brains of living patients with AD Enters the brain in sufficient amounts Stable in vivo Moderately lipophilic Low uptake of metabolites to brain Retention in the brain Detection of small amount of A High specificity for amyloid deposits Low non-specific binding
Figure 4. PET sections through the basal ganglia (left) and cerebral cortex (right) of the brain of a female carrier of the Swedish mutation given a tracer dose of 18F-fluorodeoxyglucose. The PET studies were done before cognitive symptoms and 40 months later when cognitive symptoms were present. The colour scale indicates regional cerebral glucose metabolism (rCMRglc; mol/min/100 g): red=high, yellow=medium, and blue=low. Impaired rCMRglc was observed in the parietal cortex at first scan and a progression in the rCMRglc deficits was observed at second scan in the parietal, temporal, and frontal cortices.
in vitro, neuritic plaques were observed in the hippocampus of patients.58 The plaques were visualised as areas of decreased signal intensity of T2* weighted MRM images. The accelerated T2* relaxation was caused by the presence of metal ions in the plaque. Because imaging time was 20 h the technique is probably not suitable for in vivo imaging in humans.58 Other researchers using MRM were unable to detect A plaques at autopsy of AD brain tissue.59 By injection of putrescine–gadolinium–A peptide (PUT-Gd–A) intravenously into transgenic APP mice, labelling of amyloid plaques was measured by MRI in cortical tissue ex vivo.43 Wadghiri and co-workers44 labelled A1–40 peptide with either magnetically labelled compounds gadolinium-diethylenetriaminepenta-acetic acid (Gd-DTPA) or monocrystalline iron oxide nanoparticles (MION) to detect by µMRI amyloid plaques in transgenic APP and APP/PS mice in vivo. The magnetically labelled A1–40 had to be injected with mannitol to facilitate crossing of the blood–brain barrier.44 A significant
correlation was observed between the density of amyloid plaques in brains measured by MRI and the histologically confirmed number of plaques. MION–A1–40 showed somewhat lower sensitivity to amyloid plaques than did GdDTPA–A1–40, which might be due to low permeability of the blood–brain barrier for MION–A1–40.44 The next step is for magnetically labelled A1–40 compounds to be tested in patients with AD. Small molecular methods of amyloid imaging
Several research groups have used the small-molecule approach to the development of substances suitable for amyloid imaging (figure 5). Some of the most promising compounds have been Congo red, thioflavin, stilbene, and FDDNP. The substances differ in binding characteristics and in their brain uptake. Encouraging in vitro and in vivo properties for some of the substances has recently led to promising in vivo imaging of amyloid in patients with AD.37,38 Derivatives of Congo red
Chrysamine-G is a Congo-red derivative (figure 5) with better permeability to the brain than Congo red.60 When incubated with human post-mortem brain-tissue homogenates, carbon-14-labelled chrysamine-G showed labelling of amyloid angiography and significantly higher binding in the frontal, temporal, and parietal cortices of patients with AD in comparison with those of age-matched controls.61 No significant difference in binding was observed when cerebellum was used as an internal Brains that, in histpopathological standard.61 investigations, had shown many neuritic plaques also showed 14C-chrysamine-G binding. X-34 is a lipophilic, highly fluorCompounds used for imaging of amyloid in the brains of living organisms escent derivative of Congo red and has shown promising staining Reference Imaging compound Imaging technique Study properties of the beta sheet structures Friedland et al Tc-10H3 SPECT Patients with AD of amyloid plaques and cerebroF-FDDNP PET Patients with AD Shoghi-Jadid et al vascular amyloid in AD autopsy C-PIB PET Patients with AD Klunk et al brain tissue.62 In vivo labelling of Mathis et al C-BTA-1 PET Baboons fibrillar amyloid deposits by X-34 MION-A MRI Mice PS Poduslo et al was shown in transgenic Poduslo et al PUT-Gd-A MRI Mice PS Caenorhabditis elegans.63 Gd-DTPA-A mMRI Mice APP/PS Wadghiri et al 125 I-IMSB and 125I-ISB bind with BTA-1 Multiphoton Mice APP/PS Mathis et al high affinity to human brains in Thioflavin-S Multiphoton Mice APP Bacskai et al vitro.64 The penetration to the Bacskai et al PIB Multiphoton Mice APP brain was low although detectable for 40
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PET imaging of amyloid
NH2
H2N
N
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HO
N
N NaO3S
O O
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X-34 125I
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BTA-1 Figure 5. Chemical structures of compounds used for development of in vivo amyloid imaging ligands.
I-IMSB in transgenic mice. The styrylbenzene ligand I-3 has also shown significant labelling of plaques in vitro and detect both A1–40 and A1–42.65 Like X-34, 125I-IMSB is a styrylbenzene derivative, which preferentially detects A1–40 and could be a promising SPECT ligand but unfortunately its penetration of the brain is ten times less than that of thioflavine derivatives.65 Methoxy-X04, a derivative of Congo red and chrysamine-G, is a more lipophilic compound and has higher binding affinity to A fibrils (26 nmolar).66 In mice intravenously injected with 11C-methoxy-X04 uptake to brain was high. Multiphoton imaging technology is an interesting technique because it allows visualisation of small brain structures (1 m).46 Investigation by this technique can be done in living anaesthetised animals in which a section of the skull is replaced by a round coverslip and the entry to brain of intravenously injected fluorescence compound is imaged with muliphoton microscopy. Studies by Bacskai and co-workers47 showed that this technique can be used to reveal labelling of amyloid deposits in vivo in transgenic APP mice (figure 6). 125 125
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After intravenous injection, methoxy-XO4 crosses the blood–brain barrier and binds to dense-core amyloid plaques and cerebrovascular amyloid angiopathy. Thioflavin-T derivatives
Thioflavin S and thioflavin T are amyloid-binding dyes. Thioflavin has been used as a pharmacophore for development of amyloid-binding compounds.42,45,67 Some of the synthesised derivatives of thioflavin—such as 125I-6,68 3 H-BTA-1,69 and 11C-6-OH-BTA-144—have promising binding properties in autopsy brain tissue from patients with AD with binding affinities in the nanomolar range, ten to 100 times higher than chrysamine-G. The binding of hydrogen-3-BTA-1 to neurofibrillary tangles was low in comparison with binding to both amyloid plaques and cerebrovascular amyloid.69 The binding affinity of 3 H-BTA-1 was similar in brain homogenates from patients with AD and in Aβ1–40 fibrils, which supports the assumption that 3H-BTA-1 mainly is binding to deposits of fibrillar Aβ.69 In vitro binding studies with 11C-OH-BTA-1 (PIB), frontal cortical brain tissue taken from the brains of
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Figure 6. Multiphoton microscopy of senile plaques in the brain of a live 21-month-old transgenic APP mouse (Tg 2576). Amyloid deposits were labelled by injection of 1 mg/kg methoxy-XO4 intraperitoneally 24 h before imaging (red). The anaesthetised mouse was prepared for imaging by replacing an 8 mm circular area of the skull with a glass that was cemented in place. Fluorescein was injected intravenously to label all blood vessels (green). A three-dimensional volume was acquired by collecting a series of two-dimensional images 400 m deep from the surface of the brain, and projected onto a single plane. Dense core plaques were clearly labelled with methoxy-XO4 , as well as some cerebrovascular amyloid angiopathy. Scale bar=50 m. Courtesy of Dr Brian Bacskai, Department of Neurology, Massachusetts General Hospital, Charlestown, USA.
patients with AD at autopsy showed maximum binding values similar to earlier biochemically measured amounts of A in human frontal cortex.18 These observations suggest binding of the compound to amyloid is close to one to one in human brain tissue.42 The binding of 11C-PIB was displaced by an excess of BTA-1 suggesting a competitive binding.39 Multiphoton microscopy in live transgenic mice shows fluorescent PIB crossing the blood–brain barrier, labelling amyloid deposits, and then clearing from the brain.47 125 I-TZDM is also developed from thioflavin-T and has shown higher binding affinity to synthetic A1–40 (0·06 nmolar)70,71 than the compounds studied so far. Whether the 125I-TZDM is a future amyloid ligand for SPECT studies is still an open question. Kung and co-workers72 recently reported a new thioflavin derivative 125I-IMPY, which labels amyloid plaques in transgenic mice and therefore also might be a potential amyloid ligand for SPECT. Stilbene derivatives
Stilbene derivatives have been synthesised as compounds for the probing of amyloid plaques.73 Stilbene shows binding to A aggregates in the nanomolar range.74 The stilbene derivative 11C-4 is a moderately lipophilic compound that has good penetration of brain tissue and labels amyloid plaques in trangenic mice.75 With these binding properties, stilbene derivatives should be tested for PET imaging.
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Figure 7. Serial planes showing the topography of 11C-PIB retention in the brain of a control individual and a patient with AD. Top two rows show axial and bottom two show sagittal standardised uptake values (SUV) of PIB images. Images from the the control individual are shown in rows one and three and the corresponding data from the patient with AD are shown in rows two and four. The scale bar indicates relative levels of PIB SUV values. Reproduced with permission from Wiley-Liss Inc.39
Acridine analogues
Acridine orange and analogues have been tested as amyloid ligands. Although acridine orange had low affinity, a derivative BF-108 showed high affinity to A aggregates, neuritic plaques, neurofibrillary tangles, amyloid laden vessels in autopsy brain tissue, and amyloid plaques in transgenic mice.76 DDNP analogues
F-FDDNP is a radiofluorinated 6-dialakylamino-2naphthylidene derivative of DDNP that binds to synthetic A1–40 at two binding sites, a high-affinity site (0·12 nmolar) 18
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PET imaging of amyloid
and a low-affinity site (1·9 nmolar).77 Binding studies in human autopsy brain tissue revealed binding affinity of FDDNP in the nanomolar range.11 18F-DDNP also binds to neurofibrillary tangles77,78 and to prion plaques79 in human autopsy brain tissue
Amyloid imaging in AD with 18F-FDDNP The 18F-FDDNP studies by Shoghi-Jadid and co-workers41 were the second attempt in patients with AD to detect in vivo abnormal amyloid deposition in the brain. 18F-FDDNP was given intravenously to nine patients with different degrees of cognitive impairment and seven age-matched controls. The retention of 18F-FDDNP in the temporal, parietal, frontal, and occipital cortical regions of the patients was 10–15% higher than in the pons.41 The highest retention of 18F-FDDNP in the patients was observed in the hippocampus, amygdala, and entorhinal cortex where the retention was 30% higher than in the pons. A negative correlation was observed between binding of FDDNP and cognitive status of the patients with AD.49 Binding data of FDDNP was expressed as the relative residence time.41 The data varied, however, between different regions, such as the temporal cortex and occipital cortex, despite similar equilibrium data.47 These observations might be explained by the blood-flow dependence of 18F-FDDP as well as the highly lipophilic nature of the compound.
Amyloid binding in AD with 11C-PIB The first human study with C-PIB in 16 patients with mild AD and nine healthy people has recently been published.39 When 11C-PIB was injected intravenously in a bolus dose we found that the compound rapidly reached the brain and no metabolites of PIB was produced that crossed the blood–brain barrier.39 11C-PIB showed a rapid uptake to brain and a substantial retention in the frontal, temporal, parietal, and occipital cortices and the striatum but low entry into the cerebellum and subcortical white matter of the patients.39 A rapid entry and clearance of 11C-PIB was observed in cortical and subcortical grey matter, including the cerebellar cortex of healthy people.39 Thus a slower washout of the compound from brain regions was observed in patients with mild AD than in control individuals. A robust difference was thus observed between the retention pattern in AD patients and that in healthy controls in brain regions in which amyloid deposits accumulate (figure 7).39 The retention in patients was most prominent in the frontal cortex (1·9 times that in controls) but also high in other cortical regions such as parietal, occipital, and temporal cortices (1·5–1·7 times). The high retention of PIB in the frontal cortex conflicts with evidence from post-mortem studies, in which amyloid load is rarely highest in the frontal cortex. Amyloid processing (figure 2) might differ in vivo compared with paraffin-fixed autopsy brain tissue, as might pH conditions. The influence of cerebral blood flow on the retention pattern of PIB should be assessed further. No difference was observed in PIB retention between young and healthy old people although the oldest control (77 years) showed PIB retention typical of a patient with AD.39 This finding stimulated further PIB studies in healthy old 11
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Figure 8. Top: 11C-PIB standardised uptake value (SUV) images showing a marked difference between the 11C-PIB retention in the brain of a 67-yearold healthy person (left) and a 79-year–old AD patient (right). Bottom: 18 F-FDG cerebral regional glucose metabolism (rCMRglc; mol/min/100 ml) images . A high retention of PIB was observed in the frontal and tempoparietal cortices of the patients with AD and hypometabolism in rCMRglc. Lack of PIB retention (top left) and normal rCMRglc (bottom left) was observed in the healthy person. Reproduced with permission from Wiley-Liss Inc.39
people as signs for possible preclinical AD.80 The retention of PIB in cortical AD brain regions was inversely related to the cerebral glucose metabolism as measured by 18 F-fluorodeoxyglucose in the same brain regions (figure 8).39 11 C-PIB did not show any significant correlation between amyloid load and cognitive impairments when measured as MMSE.39 Although a correlation between cognition and amyloid is not expected, further studies with more patients with varied cognitive statuses from mild to severe AD have to be done to study this topic further. The studies with 11C-PIB are promising and the potential application might be substantial.37
Future prospects The rapid development of different compounds suitable for the visualising of amyloid during the past 10 years has led to the first promising in vivo studies of the amyloid ligands PIB62 and FDDNP;41 the latter compound also seems to label neurofibrillary tangles in patients with AD. The development of several of the amyloid ligand candidates has failed because of poor passage across the blood–brain barrier or low measurable signal activity. The robust difference in PIB binding between AD tissue and control tissue creates numerous challenging questions that will inspire new PET studies. Important issues to be addressed in future studies include time course of progression of amyloid deposits in the brains of patients with hereditary and sporadic forms of AD, the presence of amyloid in the brains of patients with MCI at high risk of AD, and the follow-up of patients with AD already assessed by PIB. Further imaging studies must also focus on the binding properties of ligands to soluble and insoluble amyloid
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PET imaging of amyloid
Search strategy and selection criteria The starting point for this review article was the recent publications of original articles on the development of compounds for the imaging of amyloid in the brains of patients with AD. Additional references for this review were obtained by searches (1988–2004) of PubMed with the terms “amyloid binding”, “brain”, “Alzheimer’s disease”, and “imaging”. Only papers published in English were reviewed.
before plaque formation. These studies together with attempts to label tangles might provide further knowledge of whether the initial formation of tangles is dependent or independent of amyloid formation. The measurement of amyloid in vivo will not only have a large effect on the understanding of the underlying pathophysiological mechanisms of AD but also should aid in the testing of new antiamyloid drugs. It can be used complimentary to cognitive testings, other imaging studies including MRI, References 1 2 3 4 5 6
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and measurement of various other biological markers. The economic impact of a new imaging probe is difficult to estimate. PET imaging in the diagnosis and follow-up of cancer has shown some economical value. Further development of amyloid probes applicable for both PET and SPECT in AD may show similar economic value especially when effective antiamyloid therapy is available. Acknowledgments
Dr Zhizhong Guan for providing immunostaining amyloid data in post-mortem Alzheimer brain tissue, Mrs Marianne Grip for her professional help with the illustrations, and Dr Anders Wall for help with fluorodeoxyglucose PET images. Conflict of interest
I have no conflict of interest. Role of the funding source
AN’s research is supported by grants from the Swedish Medical Research Council, Foundation for Old Servants, Stohnes Foundation, Swedish Alzheimer Foundation, KI Foundations, Stockholm City Council. None of these funding sources had any role in the preparation of this review or in the decision to submit it for publication.
Correlation between elevated levels of amyloid betapeptide in the brain and cognitive decline. JAMA 2000; 283: 1571–77. Parvathy S, Davies P, Haroutunian V, et al. Correlation between Ax-40-, Ax-42-, and Ax43-containing amyloid plaques and cognitive decline. Arch Neurol 2001; 58: 2025–32. Selkoe DJ. Deciphering the genesis and fate of amyloid beta-protein yields novel therapies for Alzheimer disease. J Clin Invest 2002; 110: 1375–81. Cummings BJ, Cotman CW. Image analysis of betaamyloid load in Alzheimer’s disease and relation to dementia severity. Lancet 1995; 346: 1524–28. Schott JM, Fox NC, Frost C, et al. Assessing the onset of structural change in familial Alzheimer’s disease. Ann Neurol 2003; 53: 181–88. Kennedy AM, Frackowiak RS, Newman SK, et al. Deficits in cerebral glucose metabolism demonstrated by positron emission tomography in individuals at risk of familial Alzheimer’s disease. Neurosci Lett 1995; 186: 17–20. Nordberg A, Viitanen M, Almkvist O, et al. Longitudinal positron emission tomographic studies in families with chromosome 14 and 21 encoded Alzheimer’s disease. In: Iqbal K, Mortimer J, Winblad H, eds. Research advances in Alzheimer’s disease and related disorders. Chichester: John Wiley & Sons, 1995: 243–49. Wahlund LO, Basun H, Almkvist O, et al. A followup study of the family with the Swedish APP 670/671 Alzheimer’s disease mutation. Dement Geriatr Cogn Disord 1999; 10: 526–33. Small GW, Ercoli LM, Silverman DH, et al. Cerebral metabolic and cognitive decline in persons at genetic risk for Alzheimer’s disease. Proc Natl Acad Sci USA 2000; 97: 6037–42. Reiman EM, Caselli RJ, Chen K, Alexander GE, Bandy D, Frost J. Declining brain activity in cognitively normal apolipoprotein E epsilon 4 heterozygotes: a foundation for using positron emission tomography to efficiently test treatments to prevent Alzheimer’s disease. Proc Natl Acad Sci USA 2001; 98: 3334–39. Reiman EM, Chen K, Alexander GE, et al. Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer’s dementia. Proc Natl Acad Sci USA 2004; 101: 284–89. Petersen RC, Smith GE, Waring SC, Ivnik RJ, Tangalos EG, Kokmen E. Mild cognitive impairment: clinical characterization and outcome. Arch Neurol 1999; 56: 303–08. Grundman M, Petersen RC, Ferris SH, et al. Mild cognitive impairment can be distinguished from Alzheimer disease and normal aging for clinical trials. Arch Neurol 2004; 61: 59–66. Arnaiz E, Jelic V, Almkvist O, et al. Impaired cerebral glucose metabolism and cognitive functioning predict deterioration in mild cognitive impairment. Neuroreport 2001; 12: 851–55. Chetelat G, Desgranges B, de la Sayette V, Viader F, Eustache F, Baron JC. Mild cognitive impairment: can FDG-PET predict who is to rapidly convert to
Alzheimer’s disease? Neurology 2003; 60: 1374–77. 33 Wolf H, Jelic V, Gertz HJ, Nordberg A, Julin P, Wahlund LO. A critical discussion of the role of neuroimaging in mild cognitive impairment. Acta Neurol Scand Suppl 2003; 179: 52–76. 34 DeKosky ST, Marek K. Looking backward to move forward: early detection of neurodegenerative disorders. Science 2003; 302: 830–34. 35 Nordberg A. Toward an early diagnosis and treatment of Alzheimer’s disease. Int Psychogeriatr 2003; 15: 223–37. 36 Nordberg A. Functional studies of cholinergic activity in normal and Alzheimer disease states by imaging technique. Prog Brain Res 2004; 145: 301–10. 37 Jagust W. The proteomics of positron emission tomography. Ann Neurol 2004; 55: 303–05. 38 Klunk WE, Engler H, Nordberg A, et al. Imaging the pathology of Alzheimer’s disease: amyloid-imaging with positron emission tomography. In: Cidis Meltzer C, ed. Neurologic applications of positron emission tomography. Philadelphia: W Saunders Company, 2003: 781–89. 39 Klunk WE, Engler H, Nordberg A, et al. Imaging brain amyloid in Alzheimer’s disease with Pittsburgh Compound-B. Ann Neurol 2004; 55: 306–19. 40 Friedland RP, Kalaria R, Berridge M, et al. Neuroimaging of vessel amyloid in Alzheimer’s disease. Ann NY Acad Sci 1997; 826: 242–47. 41 Shoghi-Jadid K, Small GW, Agdeppa ED, et al. Localization of neurofibrillary tangles and betaamyloid plaques in the brains of living patients with Alzheimer disease. Am J Geriatr Psychiatry 2002; 10: 24–35. 42 Mathis CA, Wang Y, Holt DP, Huang GF, Debnath ML, Klunk WE. Synthesis and evaluation of 11C-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents. J Med Chem 2003; 46: 2740–54. 43 Poduslo JF, Wengenack TM, Curran GL, et al. Molecular targeting of Alzheimer’s amyloid plaques for contrast-enhanced magnetic resonance imaging. Neurobiol Dis 2002; 11: 315–29. 44 Wadghiri YZ, Sigurdsson EM, Sadowski M, et al. Detection of Alzheimer’s amyloid in transgenic mice using magnetic resonance microimaging. Magn Reson Med 2003; 50: 293–302. 45 Mathis CA, Bacskai BJ, Kajdasz ST, et al. A lipophilic thioflavin-T derivative for positron emission tomography (PET) imaging of amyloid in brain. Bioorg Med Chem Lett 2002; 12: 295–98. 46 Bacskai BJ, Kajdasz ST, Christie RH, et al. Imaging of amyloid-beta deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy. Nat Med 2001; 7: 369–72. 47 Bacskai BJ, Hickey GA, Skoch J, et al. Fourdimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-beta ligand in transgenic mice. Proc Natl Acad Sci USA 2003; 100: 12462–67. 48 Maggio JE, Stimson ER, Ghilardi JR, et al. Reversible
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in vitro growth of Alzheimer disease beta-amyloid plaques by deposition of labeled amyloid peptide. Proc Natl Acad Sci USA 1992; 89: 5462–66. Friedland RP, Majocha RE, Reno JM, Lyle LR, Marotta CA. Development of an anti-A beta monoclonal antibody for in vivo imaging of amyloid angiopathy in Alzheimer’s disease. Mol Neurobiol 1994; 9: 107–13. Lee HJ, Zhang Y, Zhu C, Duff K, Pardridge WM. Imaging brain amyloid of Alzheimer disease in vivo in transgenic mice with an Abeta peptide radiopharmaceutical. J Cereb Blood Flow Metab 2002; 22: 223–31. Zhuang ZP, Kung MP, Wilson A, et al. Structureactivity relationship of imidazo[1,2-a]pyridines as ligands for detecting beta-amyloid plaques in the brain. J Med Chem 2003; 46: 237–43. Wengenack TM, Curran GL, Poduslo JF. Targeting alzheimer amyloid plaques in vivo. Nat Biotechnol 2000; 18: 868–72. Lovat LB, Persey MR, Madhoo S, Pepys MB, Hawkins PN. The liver in systemic amyloidosis: insights from 123I serum amyloid P component scintigraphy in 484 patients. Gut 1998; 42: 727–34. Lovat LB, O’Brien AA, Armstrong SF, et al. Scintigraphy with 123I-serum amyloid P component in Alzheimer disease. Alzheimer Dis Assoc Disord 1998; 12: 208–10. Friedland RP, Shi J, Lamanna JC, Smith MA, Perry G. Prospects for noninvasive imaging of brain amyloid beta in Alzheimer’s disease. Ann NY Acad Sci 2000; 903: 123–28. Bornebroek M, Verzijlbergen JF, Haan J, et al. Potential for imaging cerebral amyloid deposits using 123I-labelled serum amyloid P component and SPET. Nucl Med Commun 1996; 17: 929–33. Shi J, Perry G, Berridge MS, et al. Labeling of cerebral amyloid beta deposits in vivo using intranasal basic fibroblast growth factor and serum amyloid P component in mice. J Nucl Med 2002; 43: 1044–51. Benveniste H, Einstein G, Kim KR, Hulette C, Johnson GA. Detection of neuritic plaques in Alzheimer’s disease by magnetic resonance microscopy. Proc Natl Acad Sci USA 1999; 96: 14079–84. Dhenain M, Privat N, Duyckaerts C, Jacobs RE. Senile plaques do not induce susceptibility effects in
T2*-weighted MR microscopic images. NMR Biomed 2002; 15: 197–203. 60 Klunk WE, Debnath ML, Pettegrew JW. Development of small molecule probes for the betaamyloid protein of Alzheimer’s disease. Neurobiol Aging 1994; 15: 691–98. 61 Klunk WE, Debnath ML, Pettegrew JW. Chrysamine-G binding to Alzheimer and control brain: autopsy study of a new amyloid probe. Neurobiol Aging 1995; 16: 541–48. 62 Styren SD, Hamilton RL, Styren GC, Klunk WE. X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer’s disease pathology. J Histochem Cytochem 2000; 48: 1223–32. 63 Link CD, Johnson CJ, Fonte V, et al. Visualization of fibrillar amyloid deposits in living, transgenic Caenorhabditis elegans animals using the sensitive amyloid dye, X-34. Neurobiol Aging 2001; 22: 217–26. 64 Zhuang ZP, Kung MP, Hou C, et al. Radioiodinated styrylbenzenes and thioflavins as probes for amyloid aggregates. J Med Chem 2001; 44: 1905–14. 65 Kung MP, Hou C, Zhuang ZP, et al. Radioiodinated styrylbenzene derivatives as potential SPECT imaging agents for amyloid plaque detection in Alzheimer’s disease. J Mol Neurosci 2002; 19: 7–10. 66 Klunk WE, Bacskai BJ, Mathis CA, et al. Imaging Abeta plaques in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered Congo red derivative. J Neuropathol Exp Neurol 2002; 61: 797–805. 67 Klunk WE, Wang Y, Huang GF, Debnath ML, Holt DP, Mathis CA. Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain. Life Sci 2001; 69: 1471–84. 68 Wang Y, Klunk WE, Huang GF, Debnath ML, Holt DP, Mathis CA. Synthesis and evaluation of 2-(3⬘-iodo-4⬘-aminophenyl)-6-hydroxybenzothiazole for in vivo quantitation of amyloid deposits in Alzheimer’s disease. J Mol Neurosci 2002; 19: 11–16. 69 Klunk WE, Wang Y, Huang GF, et al. The binding of 2-(4⬘-methylaminophenyl) benzothiazole to postmortem brain homogenates is dominated by the amyloid component. J Neurosci 2003; 23: 2086–92.
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70 Kung MP, Zhuang ZP, Hou C, Jin LW, Kung HF. Characterization of radioiodinated ligand binding to amyloid beta plaques. J Mol Neurosci 2003; 20: 249–54. 71 Lee CW, Kung MP, Hou C, Kung HF. Dimethylamino-fluorenes: ligands for detecting beta-amyloid plaques in the brain. Nucl Med Biol 2003; 30: 573–80. 72 Kung MP, Hou C, Zhuang ZP, Cross AJ, Maier DL, Kung HF. Characterization of IMPY as a potential imaging agent for beta-amyloid plaques in double transgenic PSAPP mice. Eur J Nucl Med Mol Imaging 2004; published online March 9. 73 Kung HF, Lee CW, Zhuang ZP, Kung MP, Hou C, Plossl K. Novel stilbenes as probes for amyloid plaques. J Am Chem Soc 2001; 123: 12740–41. 74 Kung HF, Kung MP, Zhuang ZP, et al. Iodinated tracers for imaging amyloid plaques in the brain. Mol Imaging Biol 2003; 5: 418–26. 75 Ono M, Wilson A, Nobrega J, et al. 11C-labeled stilbene derivatives as Abeta-aggregate-specific PET imaging agents for Alzheimer’s disease. Nucl Med Biol 2003; 30: 565–71. 76 Suemoto T, Okamura N, Shiomitsu T, et al. In vivo labeling of amyloid with BF-108. Neurosci Res 2004; 48: 65–74. 77 Agdeppa ED, Kepe V, Liu J, et al. Binding characteristics of radiofluorinated 6-dialkylamino-2naphthylethylidene derivatives as positron emission tomography imaging probes for beta-amyloid plaques in Alzheimer’s disease. J Neurosci 2001; 21: RC189. 78 Agdeppa ED, Kepe V, Liu J, et al. 2-Dialkylamino-6acylmalononitrile substituted naphthalenes (DDNP analogs): novel diagnostic and therapeutic tools in Alzheimer’s disease. Mol Imaging Biol 2003; 5: 404–17. 79 Bresjanac M, Smid LM, Vovko TD, Petric A, Barrio JR, Popovic M. Molecular-imaging probe 2-(1-[6-[(2-fluoroethyl)(methyl) amino]-2naphthyl]ethylidene) malononitrile labels prion plaques in vitro. J Neurosci 2003; 23: 8029–33. 80 Schmitt FA, Davis DG, Wekstein DR, Smith CD, Ashford JW, Markesbery WR. “Preclinical” AD revisited: neuropathology of cognitively normal older adults. Neurology 2000; 55: 370–76.
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Agneta Nordberg Alzheimer’s disease (AD) is the most common form of dementia and is characterised by progressive impairment in cognitive function and behaviour. The pathological features of AD include neuritic plaques composed of amyloid- peptide (A) fibrils, neurofibrillary tangles of hyperphosphorylated tau, and neurotransmitter deficits. Increases in the concentration of A in the course of the disease with subtle effects on synaptic efficacy will lead to gradual increase in the load of amyloid plaques and progression in cognitive impairment. Direct imaging of amyloid load in patients with AD in vivo would be very useful for the early diagnosis of AD and the development and assessment of new treatment strategies. Three different strategies are being used to develop compounds suitable for in vivo imaging of amyloid deposits in human brains. Monoclonal antibodies against A and peptide fragments have had limited uptake by the brain when tested in patients with AD. When putrescine-gadolinium-A has been injected into transgenic mice overexpressing amyloid, labelling has been observed with MRI. The small molecular approach for amyloid imaging has so far been most successful. The binding of different derivatives of Congo red and thioflavin has been studied in human autopsy brain tissue and in transgenic mice. Two compounds, fluorine-18-labelledFDDNP and carbon-11-labelled-PIB, both show more binding in the brains of patients with AD than in those of healthy people. Additional compounds will probably be developed that are suitable not only for PET but also for single photon emission CT (SPECT). Lancet Neurol 2004; 3: 519–27
Alzheimer’s disease (AD) accounts for 60–70% of all dementia and is characterised by irreversible memory impairment, continuous cognitive decline, and behavioural disturbances. The duration of the disease can vary between 5 years and 20 years. About 6% of the population of age over 65 years have dementia and the number of new cases in 2000 was estimated to be 4·6 million worldwide.1 Because of the increase in the number of elderly people, it is estimated that the worldwide number of patients with dementia (25 million in 2000) will increase to 63 million in 2030 and to 114 million in 2050.1 The increasing number of people with dementia is a great challenge for society and health-care systems. Although there has been a rapid increase in the understanding of the aetiology, genetics, and underlying pathophysiological mechanism for AD during recent years there is still no cure for the disease. Therapy is mainly symptomatic and great efforts are made to develop new
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drugs that lower amyloid load in the brain and that have neuroprotective properties to slow disease progression. Early detection of AD will be a prerequisite for early treatment. Several compounds have been developed for the imaging of amyloid: radiolabelled amyloid- peptide (A) antibodies and peptide fragments; small molecules (such as derivatives of Congo red, thioflavin, stilbene, and acridine) for PET and SPECT imaging; and compounds for MRI. The aim of this review is to describe the search for and development of amyloid imaging compounds, which, after studies in post-mortem human brain tissue and transgenic APP mice, finally led to PET studies in patients. Although there are limitations to amyloid-imaging technology, and further validation and development is needed for routine clinical assessment, imaging should be used to assess the effectiveness of antiamyloid therapy.
The amyloid hypothesis The presence of amyloid in a form of dementia (which later became known as AD) was described by Alois Alzheimer.2 The German physician, described the presence of amyloid plaques and neurofibrillary tangles in the brain of a 51-yearold woman named Auguste D who had a history of progressive memory impairment.3 The extracellular plaques and deposits and intracellular neurofibrillary tangles became the hallmark pathological features of AD together with neuronal and synaptic losses and neurotransmitter deficits.4,5 Purified and sequenced by Glenner and Wong in 1984,6 amyloid is present in plaques as small insoluble A peptides.7 Amyloid deposits can be morphologically classified into neuritic plaques, amyloid angiopathy of the capillaries, amyloid angiopathy of the arteries and veins, and diffuse amyloid deposits.8 The depositing of A may be an early and obligatory event in the pathogenesis of AD.9 A gradual cortical depositing of amyloid was described by Braak and Braak.9 They proposed that amyloid deposits first appeared in the basal neocortex, and spread to all areas of the cortex (figure 1);9 at later stages the deposits also involved subcortical brain regions, including the
AN is at the Karolinska institute, Neurotec Department, Division of Molecular Neuropharmacology, Karolinska University Hospital Huddinge, Stockholm, Sweden. Correspondence: Prof Agneta Nordberg, Karolinska institute, Neurotec Department, Division of Molecular Neuropharmacology and Department of Geriatric Medicine, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. Tel +46 8 58585467; fax +46 8 6899210; email [email protected]
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impairment of brain glucose metabolism at presymptomatic stages years before clinical symptoms.23–25 My colleagues and I have recently observed impairment in cerebral glucose metabolism in presymptomatic carriers of a PS1 mutation, 10–15 years before estimated onset of AD (unpublished). Progressive impairment of cerebral glucose metabolism in people with the Swedish mutation can be visualised with PET (figure 4). People who have the (APOE) ⑀4 allele have low rates of cerebral glucose metabolism in their brains decades before a possible onset of AD.26–28 Early abnormal patterns of glucose metabolism in people at risk of AD highlight the importance of identifying AD at a Figure 1. Evolution of amyloid deposits in AD. From the analysis of the presence of amyloid in presymptomatic stage. brains obtained at autopsy three stages can be distinguished in the gradual development of cortical amyloid deposition in AD. Courtesy of Dr Heikki Braak, Department of Pathology, People with mild cognitive Frankfurt University, Frankfurt, Germany. impairment (MCI) have a high risk of AD.29 MCI is an intermediate stage cerebellum.10 The distribution of plaques differs from that of between cognitive change with normal ageing and what neurofibrillary tangles.11 The finding of genetic mutations might constitute an early stage of AD. The distinction that cause AD by affecting the processing of amyloid between normal ageing and MCI might be quite subtle. precursor protein (APP) led to the amyloid cascade Compared with control individuals, people with MCI have hypothesis12 with increased production of A1–42, A1–40, impairments in memory function and are more likely to amyloid fibrils, and amyloid plaques (figure 2). Amyloid carry the APOE ⑀4 allele.30 Morphological and glucose pathology is assumed to precede the formation of tau- metabolism changes are found in patients with MCI who positive paired helical filaments (neurofibrillary tangles), will develop AD.31–33 These people also have amyloid pathogenetic microglial processes, and oxidative stress pathology in higher amounts than expected for age at reactions.13 The APP 670/671 (Swedish) mutation14 autopsy but have preserved cholinergic enzymes.34 In order produced highly aggregable forms of A1–42 and more and to understand and detect early AD, not only functional larger plaques than in sporadic AD15–17 (figure 3). A crucial consequences of the pathological processes but also in vivo question is whether soluble or insoluble oligomers or mature neurochemistry and imaging of aetiological and pathological amyloids are most neurotoxic (figure 2). Measurement of processes including A pathology need to be measured.34–39 A concentrations in post-mortem brain tissue revealed The possibility of imaging and quantifying amyloid in high concentrations of A1–42 and A1–40 in early stage of living patients with AD will enable a critical assessment of dementia associated with cognitive decline.18,19 Subtle the amyloid hypothesis. Amyloid imaging as a surrogate changes in soluble oligomers of A might lead to synaptic marker will provide presymptomatic detection and allow dysfunction in the brains of patients with AD.20 The amount studies of disease progression. Amyloid imaging in people of amyloid deposits may not be of relevance for status of from families with hereditary forms of AD including cognition although a strong association has been reported chromosomal aberrations (APP and PS1 mutations) between amyloid load in entorhinal cortex and cognition.21 as well as carriers of APOE ⑀4 is therefore of highest Studies to assess the benefits of antiamyloid therapy, the priority. Ongoing studies with antiamyloid therapy, such as visualisation of amyloid load, and its association with vaccination, have shown some difficulties in the assessment cognitive effects are needed. of drug effects at autopsy. The visualisation of amyloid plaques in the brains of living patients with AD would Early detection of AD greatly aid the assessment of efficacy for antiamyloid The rapid recent development of non-invasive tools for the therapy. imaging of human brains has had a great effect on our ability to investigate and understand brain function. Structural Amyloid imaging brain imaging, such as MRI as well as functional brain An ideal imaging ligand should fulfil several criteria (panel). imaging with single photon emission CT (SPECT) and PET In the past 10 years several different strategies have been have revealed and increased the understanding of early used to develop compounds suitable for amyloid imaging changes in AD. Longitudinal studies in families with AD with different chemical structures and properties (figure 5). caused by mutations in the APP and presenilin genes The binding properties of the compounds have been studied have shown evidence for structural changes22 and in vitro by autoradiographic ligand studies with tissue
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APP N APPs
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Figure 3. Neuritic plaques and astrocytes visualised by immunostaining with antibodies against A and GFAP in thin tissue paraffin fixed autopsy AD brain tissue. More and larger neuritic plaques were observed in the temporal cerebral cortex of a 62-year-old woman with the Swedish mutation14 than in a 66-year-old man with sporadic AD (inserted figure). More astrocytes were distributed around the amyloid plaques in the brain of the patient with genetic AD.
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Figure 2. APP processing pathways and formation of A peptide. APP is initially cleaved by ␣ and  secretases, generating the soluble fragments ␣APP and APPs and the C-terminal fragments C83 and C99. Further ␥−secretase processing of the C-terminal fragments releases p3 and A respectively. A protofibrils have a secondary structure rich in -sheets that can give rise to the amyloid fibrils present in plaques.
homogenates or thin slices from mice or human brains. Labelled compounds have been injected into mice to study in vivo uptake and binding. Some promising compounds have been visualised with different imaging techniques in mice, monkeys, and patients with AD (table).39–47 The development of plaque-binding compounds started with monoclonal antibodies against A and peptide fragments,48–50 which were followed by small radiolabelled analogues of Congo red, chrysamine-G, and thioflavin42,51 applicable for SPECT and PET, and molecules targeting amyloid plaques suitable for MRI.43,44 The compounds fluorine-18-labelled FDDNP41 and the Pittsburgh Compound-B (carbon-11-labelled PIB)39 have both been given to human beings and the uptake and binding in brain have been studied by PET in patients with AD (table).
temporal cortex was observed.48 Antibodies to A1–28 labelled with technetium-99 were developed by Friedland and co-workers.49 The monoclonal antibody 10H3 was identified as a promising amyloid imaging agent.40 When the compound was given to six people with probable AD, SPECT studies revealed an uptake of 99Tc-10H3 solely around the scalp and cranial bone marrow, whereas no cerebral uptake was observed.40 In order to increase the brain permeability to A1–40, Wengenack and co-workers52 used putrescine modified A1–40, which showed higher permeability to brain than the natural peptide and labelled amyloid deposits in transgenic APP/PS mice.52 Similarly, when a conjugation of iodine-125-labelled A1–40 to 8D3 monoclonal antibody was used, uptake of the peptide and binding to A was observed in the brain of transgenic mice.50 Lovat and co-workers53 imaged amyloid in peripheral tissue of patients with systemic amyloidosis by use of iodine-123-labelled serum amyloid P component (SAP) and SPECT. The same ligand was used for imaging of amyloid in the brains of patients with AD. No binding of 123I-SAP was observed,54 probably because of poor passage across the blood–brain barrier.55 However, Bornebroek and co-workers56 reported accumulation of 123I-SAP in the cerebral cortex when it was given to two patients with hereditary cerebral amyloid angiopathy (Dutch type), which was imaged with SPECT. Labelling of cerebral amyloid has been observed in transgenic APP mice after intranasal injection of 125I-basic fibroblast growth factor (125I-bFGF) and SAP.54 The authors suggest that intranasal injection of radiolabelled compounds might be promising for the imaging of brain amyloid in human beings.57 Detection of brain amyloid by MRI
Radiolabelled A antibodies and peptide fragments
Antibodies to A have been tested as suitable imaging agents for amyloid. A saturable binding of iodine-125-labelled A1–40 protein to tissue homogenates prepared from AD
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Magnetic resonance microscopy (MRM) produces images with higher spatial resolution than conventional MRI. When MRM was applied in human brain autopsy tissue from five patients with AD and three age-matched control individuals
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Criteria for an ideal imaging compound for the detection of amyloid in the brains of living patients with AD Enters the brain in sufficient amounts Stable in vivo Moderately lipophilic Low uptake of metabolites to brain Retention in the brain Detection of small amount of A High specificity for amyloid deposits Low non-specific binding
Figure 4. PET sections through the basal ganglia (left) and cerebral cortex (right) of the brain of a female carrier of the Swedish mutation given a tracer dose of 18F-fluorodeoxyglucose. The PET studies were done before cognitive symptoms and 40 months later when cognitive symptoms were present. The colour scale indicates regional cerebral glucose metabolism (rCMRglc; mol/min/100 g): red=high, yellow=medium, and blue=low. Impaired rCMRglc was observed in the parietal cortex at first scan and a progression in the rCMRglc deficits was observed at second scan in the parietal, temporal, and frontal cortices.
in vitro, neuritic plaques were observed in the hippocampus of patients.58 The plaques were visualised as areas of decreased signal intensity of T2* weighted MRM images. The accelerated T2* relaxation was caused by the presence of metal ions in the plaque. Because imaging time was 20 h the technique is probably not suitable for in vivo imaging in humans.58 Other researchers using MRM were unable to detect A plaques at autopsy of AD brain tissue.59 By injection of putrescine–gadolinium–A peptide (PUT-Gd–A) intravenously into transgenic APP mice, labelling of amyloid plaques was measured by MRI in cortical tissue ex vivo.43 Wadghiri and co-workers44 labelled A1–40 peptide with either magnetically labelled compounds gadolinium-diethylenetriaminepenta-acetic acid (Gd-DTPA) or monocrystalline iron oxide nanoparticles (MION) to detect by µMRI amyloid plaques in transgenic APP and APP/PS mice in vivo. The magnetically labelled A1–40 had to be injected with mannitol to facilitate crossing of the blood–brain barrier.44 A significant
correlation was observed between the density of amyloid plaques in brains measured by MRI and the histologically confirmed number of plaques. MION–A1–40 showed somewhat lower sensitivity to amyloid plaques than did GdDTPA–A1–40, which might be due to low permeability of the blood–brain barrier for MION–A1–40.44 The next step is for magnetically labelled A1–40 compounds to be tested in patients with AD. Small molecular methods of amyloid imaging
Several research groups have used the small-molecule approach to the development of substances suitable for amyloid imaging (figure 5). Some of the most promising compounds have been Congo red, thioflavin, stilbene, and FDDNP. The substances differ in binding characteristics and in their brain uptake. Encouraging in vitro and in vivo properties for some of the substances has recently led to promising in vivo imaging of amyloid in patients with AD.37,38 Derivatives of Congo red
Chrysamine-G is a Congo-red derivative (figure 5) with better permeability to the brain than Congo red.60 When incubated with human post-mortem brain-tissue homogenates, carbon-14-labelled chrysamine-G showed labelling of amyloid angiography and significantly higher binding in the frontal, temporal, and parietal cortices of patients with AD in comparison with those of age-matched controls.61 No significant difference in binding was observed when cerebellum was used as an internal Brains that, in histpopathological standard.61 investigations, had shown many neuritic plaques also showed 14C-chrysamine-G binding. X-34 is a lipophilic, highly fluorCompounds used for imaging of amyloid in the brains of living organisms escent derivative of Congo red and has shown promising staining Reference Imaging compound Imaging technique Study properties of the beta sheet structures Friedland et al Tc-10H3 SPECT Patients with AD of amyloid plaques and cerebroF-FDDNP PET Patients with AD Shoghi-Jadid et al vascular amyloid in AD autopsy C-PIB PET Patients with AD Klunk et al brain tissue.62 In vivo labelling of Mathis et al C-BTA-1 PET Baboons fibrillar amyloid deposits by X-34 MION-A MRI Mice PS Poduslo et al was shown in transgenic Poduslo et al PUT-Gd-A MRI Mice PS Caenorhabditis elegans.63 Gd-DTPA-A mMRI Mice APP/PS Wadghiri et al 125 I-IMSB and 125I-ISB bind with BTA-1 Multiphoton Mice APP/PS Mathis et al high affinity to human brains in Thioflavin-S Multiphoton Mice APP Bacskai et al vitro.64 The penetration to the Bacskai et al PIB Multiphoton Mice APP brain was low although detectable for 40
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N-methyl-11C-PIB
BTA-1 Figure 5. Chemical structures of compounds used for development of in vivo amyloid imaging ligands.
I-IMSB in transgenic mice. The styrylbenzene ligand I-3 has also shown significant labelling of plaques in vitro and detect both A1–40 and A1–42.65 Like X-34, 125I-IMSB is a styrylbenzene derivative, which preferentially detects A1–40 and could be a promising SPECT ligand but unfortunately its penetration of the brain is ten times less than that of thioflavine derivatives.65 Methoxy-X04, a derivative of Congo red and chrysamine-G, is a more lipophilic compound and has higher binding affinity to A fibrils (26 nmolar).66 In mice intravenously injected with 11C-methoxy-X04 uptake to brain was high. Multiphoton imaging technology is an interesting technique because it allows visualisation of small brain structures (1 m).46 Investigation by this technique can be done in living anaesthetised animals in which a section of the skull is replaced by a round coverslip and the entry to brain of intravenously injected fluorescence compound is imaged with muliphoton microscopy. Studies by Bacskai and co-workers47 showed that this technique can be used to reveal labelling of amyloid deposits in vivo in transgenic APP mice (figure 6). 125 125
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After intravenous injection, methoxy-XO4 crosses the blood–brain barrier and binds to dense-core amyloid plaques and cerebrovascular amyloid angiopathy. Thioflavin-T derivatives
Thioflavin S and thioflavin T are amyloid-binding dyes. Thioflavin has been used as a pharmacophore for development of amyloid-binding compounds.42,45,67 Some of the synthesised derivatives of thioflavin—such as 125I-6,68 3 H-BTA-1,69 and 11C-6-OH-BTA-144—have promising binding properties in autopsy brain tissue from patients with AD with binding affinities in the nanomolar range, ten to 100 times higher than chrysamine-G. The binding of hydrogen-3-BTA-1 to neurofibrillary tangles was low in comparison with binding to both amyloid plaques and cerebrovascular amyloid.69 The binding affinity of 3 H-BTA-1 was similar in brain homogenates from patients with AD and in Aβ1–40 fibrils, which supports the assumption that 3H-BTA-1 mainly is binding to deposits of fibrillar Aβ.69 In vitro binding studies with 11C-OH-BTA-1 (PIB), frontal cortical brain tissue taken from the brains of
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Figure 6. Multiphoton microscopy of senile plaques in the brain of a live 21-month-old transgenic APP mouse (Tg 2576). Amyloid deposits were labelled by injection of 1 mg/kg methoxy-XO4 intraperitoneally 24 h before imaging (red). The anaesthetised mouse was prepared for imaging by replacing an 8 mm circular area of the skull with a glass that was cemented in place. Fluorescein was injected intravenously to label all blood vessels (green). A three-dimensional volume was acquired by collecting a series of two-dimensional images 400 m deep from the surface of the brain, and projected onto a single plane. Dense core plaques were clearly labelled with methoxy-XO4 , as well as some cerebrovascular amyloid angiopathy. Scale bar=50 m. Courtesy of Dr Brian Bacskai, Department of Neurology, Massachusetts General Hospital, Charlestown, USA.
patients with AD at autopsy showed maximum binding values similar to earlier biochemically measured amounts of A in human frontal cortex.18 These observations suggest binding of the compound to amyloid is close to one to one in human brain tissue.42 The binding of 11C-PIB was displaced by an excess of BTA-1 suggesting a competitive binding.39 Multiphoton microscopy in live transgenic mice shows fluorescent PIB crossing the blood–brain barrier, labelling amyloid deposits, and then clearing from the brain.47 125 I-TZDM is also developed from thioflavin-T and has shown higher binding affinity to synthetic A1–40 (0·06 nmolar)70,71 than the compounds studied so far. Whether the 125I-TZDM is a future amyloid ligand for SPECT studies is still an open question. Kung and co-workers72 recently reported a new thioflavin derivative 125I-IMPY, which labels amyloid plaques in transgenic mice and therefore also might be a potential amyloid ligand for SPECT. Stilbene derivatives
Stilbene derivatives have been synthesised as compounds for the probing of amyloid plaques.73 Stilbene shows binding to A aggregates in the nanomolar range.74 The stilbene derivative 11C-4 is a moderately lipophilic compound that has good penetration of brain tissue and labels amyloid plaques in trangenic mice.75 With these binding properties, stilbene derivatives should be tested for PET imaging.
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Figure 7. Serial planes showing the topography of 11C-PIB retention in the brain of a control individual and a patient with AD. Top two rows show axial and bottom two show sagittal standardised uptake values (SUV) of PIB images. Images from the the control individual are shown in rows one and three and the corresponding data from the patient with AD are shown in rows two and four. The scale bar indicates relative levels of PIB SUV values. Reproduced with permission from Wiley-Liss Inc.39
Acridine analogues
Acridine orange and analogues have been tested as amyloid ligands. Although acridine orange had low affinity, a derivative BF-108 showed high affinity to A aggregates, neuritic plaques, neurofibrillary tangles, amyloid laden vessels in autopsy brain tissue, and amyloid plaques in transgenic mice.76 DDNP analogues
F-FDDNP is a radiofluorinated 6-dialakylamino-2naphthylidene derivative of DDNP that binds to synthetic A1–40 at two binding sites, a high-affinity site (0·12 nmolar) 18
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PET imaging of amyloid
and a low-affinity site (1·9 nmolar).77 Binding studies in human autopsy brain tissue revealed binding affinity of FDDNP in the nanomolar range.11 18F-DDNP also binds to neurofibrillary tangles77,78 and to prion plaques79 in human autopsy brain tissue
Amyloid imaging in AD with 18F-FDDNP The 18F-FDDNP studies by Shoghi-Jadid and co-workers41 were the second attempt in patients with AD to detect in vivo abnormal amyloid deposition in the brain. 18F-FDDNP was given intravenously to nine patients with different degrees of cognitive impairment and seven age-matched controls. The retention of 18F-FDDNP in the temporal, parietal, frontal, and occipital cortical regions of the patients was 10–15% higher than in the pons.41 The highest retention of 18F-FDDNP in the patients was observed in the hippocampus, amygdala, and entorhinal cortex where the retention was 30% higher than in the pons. A negative correlation was observed between binding of FDDNP and cognitive status of the patients with AD.49 Binding data of FDDNP was expressed as the relative residence time.41 The data varied, however, between different regions, such as the temporal cortex and occipital cortex, despite similar equilibrium data.47 These observations might be explained by the blood-flow dependence of 18F-FDDP as well as the highly lipophilic nature of the compound.
Amyloid binding in AD with 11C-PIB The first human study with C-PIB in 16 patients with mild AD and nine healthy people has recently been published.39 When 11C-PIB was injected intravenously in a bolus dose we found that the compound rapidly reached the brain and no metabolites of PIB was produced that crossed the blood–brain barrier.39 11C-PIB showed a rapid uptake to brain and a substantial retention in the frontal, temporal, parietal, and occipital cortices and the striatum but low entry into the cerebellum and subcortical white matter of the patients.39 A rapid entry and clearance of 11C-PIB was observed in cortical and subcortical grey matter, including the cerebellar cortex of healthy people.39 Thus a slower washout of the compound from brain regions was observed in patients with mild AD than in control individuals. A robust difference was thus observed between the retention pattern in AD patients and that in healthy controls in brain regions in which amyloid deposits accumulate (figure 7).39 The retention in patients was most prominent in the frontal cortex (1·9 times that in controls) but also high in other cortical regions such as parietal, occipital, and temporal cortices (1·5–1·7 times). The high retention of PIB in the frontal cortex conflicts with evidence from post-mortem studies, in which amyloid load is rarely highest in the frontal cortex. Amyloid processing (figure 2) might differ in vivo compared with paraffin-fixed autopsy brain tissue, as might pH conditions. The influence of cerebral blood flow on the retention pattern of PIB should be assessed further. No difference was observed in PIB retention between young and healthy old people although the oldest control (77 years) showed PIB retention typical of a patient with AD.39 This finding stimulated further PIB studies in healthy old 11
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Figure 8. Top: 11C-PIB standardised uptake value (SUV) images showing a marked difference between the 11C-PIB retention in the brain of a 67-yearold healthy person (left) and a 79-year–old AD patient (right). Bottom: 18 F-FDG cerebral regional glucose metabolism (rCMRglc; mol/min/100 ml) images . A high retention of PIB was observed in the frontal and tempoparietal cortices of the patients with AD and hypometabolism in rCMRglc. Lack of PIB retention (top left) and normal rCMRglc (bottom left) was observed in the healthy person. Reproduced with permission from Wiley-Liss Inc.39
people as signs for possible preclinical AD.80 The retention of PIB in cortical AD brain regions was inversely related to the cerebral glucose metabolism as measured by 18 F-fluorodeoxyglucose in the same brain regions (figure 8).39 11 C-PIB did not show any significant correlation between amyloid load and cognitive impairments when measured as MMSE.39 Although a correlation between cognition and amyloid is not expected, further studies with more patients with varied cognitive statuses from mild to severe AD have to be done to study this topic further. The studies with 11C-PIB are promising and the potential application might be substantial.37
Future prospects The rapid development of different compounds suitable for the visualising of amyloid during the past 10 years has led to the first promising in vivo studies of the amyloid ligands PIB62 and FDDNP;41 the latter compound also seems to label neurofibrillary tangles in patients with AD. The development of several of the amyloid ligand candidates has failed because of poor passage across the blood–brain barrier or low measurable signal activity. The robust difference in PIB binding between AD tissue and control tissue creates numerous challenging questions that will inspire new PET studies. Important issues to be addressed in future studies include time course of progression of amyloid deposits in the brains of patients with hereditary and sporadic forms of AD, the presence of amyloid in the brains of patients with MCI at high risk of AD, and the follow-up of patients with AD already assessed by PIB. Further imaging studies must also focus on the binding properties of ligands to soluble and insoluble amyloid
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PET imaging of amyloid
Search strategy and selection criteria The starting point for this review article was the recent publications of original articles on the development of compounds for the imaging of amyloid in the brains of patients with AD. Additional references for this review were obtained by searches (1988–2004) of PubMed with the terms “amyloid binding”, “brain”, “Alzheimer’s disease”, and “imaging”. Only papers published in English were reviewed.
before plaque formation. These studies together with attempts to label tangles might provide further knowledge of whether the initial formation of tangles is dependent or independent of amyloid formation. The measurement of amyloid in vivo will not only have a large effect on the understanding of the underlying pathophysiological mechanisms of AD but also should aid in the testing of new antiamyloid drugs. It can be used complimentary to cognitive testings, other imaging studies including MRI, References 1 2 3 4 5 6
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and measurement of various other biological markers. The economic impact of a new imaging probe is difficult to estimate. PET imaging in the diagnosis and follow-up of cancer has shown some economical value. Further development of amyloid probes applicable for both PET and SPECT in AD may show similar economic value especially when effective antiamyloid therapy is available. Acknowledgments
Dr Zhizhong Guan for providing immunostaining amyloid data in post-mortem Alzheimer brain tissue, Mrs Marianne Grip for her professional help with the illustrations, and Dr Anders Wall for help with fluorodeoxyglucose PET images. Conflict of interest
I have no conflict of interest. Role of the funding source
AN’s research is supported by grants from the Swedish Medical Research Council, Foundation for Old Servants, Stohnes Foundation, Swedish Alzheimer Foundation, KI Foundations, Stockholm City Council. None of these funding sources had any role in the preparation of this review or in the decision to submit it for publication.
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