Guo Feng Huang

Imaging brain amyloid in Alzheimer's disease with Pittsburgh Compound‐B

This report describes the first human study of a novel amyloid‐imaging positron emission tomography (PET) tracer, termed Pittsburgh Compound‐B (PIB), in 16 patients with diagnosed mild AD and 9 controls. Compared with controls, AD patients typically showed marked retention of PIB in areas of association cortex known to contain large amounts of amyloid deposits in AD. In the AD patient group, PIB retention was increased most prominently in frontal cortex (1.94‐fold, p = 0.0001). Large increases also were observed in parietal (1.71‐fold, p = 0.0002), temporal (1.52‐fold, p = 0.002), and occipital (1.54‐fold, p = 0.002) cortex and the striatum (1.76‐fold, p = 0.0001). PIB retention was equivalent in AD patients and controls in areas known to be relatively unaffected by amyloid deposition (such as subcortical white matter, pons, and cerebellum). Studies in three young (21 years) and six older healthy controls (69.5 ± 11 years) showed low PIB retention in cortical areas and no significant group differences between young and older controls. In cortical areas, PIB retention correlated inversely with cerebral glucose metabolism determined with 18F‐fluorodeoxyglucose. This relationship was most robust in the parietal cortex (r = −0.72; p = 0.0001). The results suggest that PET imaging with the novel tracer, PIB, can provide quantitative information on amyloid deposits in living subjects.

Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain

In vivo assessment of the beta-sheet proteins deposited in amyloid plaques (A beta peptide) or neurofibrillary tangles (tau protein) presents a target for the development of biological markers for Alzheimers disease (AD). In an effort to develop in vivo beta-sheet imaging probes, derivatives of thioflavin-T (ThT) were synthesized and evaluated. These compounds lack the positively charged quaternary heterocyclic nitrogen of ThT and are therefore uncharged at physiological pH. They are 600-fold more lipophilic than ThT. These ThT derivatives bind to A beta(1-40) fibrils with higher affinity (Ki = 20.2 nM) than ThT (Ki = 890 nM). The uncharged ThT derivatives stained both plaques and neurofibrillary tangles in post-mortem AD brain, showing some preference for plaque staining. A carbon-11 labeled compound, [N-methyl-11C]6-Me-BTA-1, was prepared, and its brain entry and clearance were studied in Swiss-Webster mice. This compound entered the brain at levels comparable to commonly used neuroreceptor imaging agents (0.223 %ID-kg/g or 7.61 %ID/g at 2 min post-injection) and showed good clearance of free and non-specifically bound radioactivity in normal rodent brain tissue (brain clearance t(1,2) = 20 min). The combination of relatively high affinity for amyloid, specificity for staining plaques and neurofibrillary tangles in post-mortem AD brain, and good brain entry and clearance makes [N-methyl-11C]6-Me-BTA-1 a promising candidate as an in vivo positron emission tomography (PET) beta-sheet imaging agent.

Binding of the Positron Emission Tomography Tracer Pittsburgh Compound-B Reflects the Amount of Amyloid-β in Alzheimer's Disease Brain But Not in Transgenic Mouse Brain

During the development of in vivo amyloid imaging agents, an effort was made to use micro-positron emission tomography (PET) imaging in the presenilin-1 (PS1)/amyloid precursor protein (APP) transgenic mouse model of CNS amyloid deposition to screen new compounds and further study Pittsburgh Compound-B (PIB), a PET tracer that has been shown to be retained well in amyloid-containing areas of Alzheimers disease (AD) brain. Unexpectedly, we saw no significant retention of PIB in this model even at 12 months of age when amyloid deposition in the PS1/APP mouse typically exceeds that seen in AD. This study describes a series of ex vivo and postmortem in vitro studies designed to explain this low retention. Ex vivo brain pharmacokinetic studies confirmed the low in vivo PIB retention observed in micro-PET experiments. In vitro binding studies showed that PS1/APP brain tissue contained less than one high-affinity (Kd = 1-2 nm) PIB binding site per 1000 molecules of amyloid-β (Aβ), whereas AD brain contained >500 PIB binding sites per 1000 molecules of Aβ. Synthetic Aβ closely resembled PS1/APP brain in having less than one high-affinity PIB binding site per 1000 molecules of Aβ, although the characteristics of the few high-affinity PIB binding sites found on synthetic Aβ were very similar to those found in AD brain. We hypothesize that differences in the time course of deposition or tissue factors present during deposition lead to differences in secondary structure between Aβ deposited in AD brain and either synthetic Aβ or Aβ deposited in PS1/APP brain.

Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-β ligand in transgenic mice

The lack of a specific biomarker makes preclinical diagnosis of Alzheimers disease (AD) impossible, and it precludes assessment of therapies aimed at preventing or reversing the course of the disease. The development of a tool that enables direct, quantitative detection of the amyloid-β deposits found in the disease would provide an excellent biomarker. This article demonstrates the real-time biodistribution kinetics of an imaging agent in transgenic mouse models of AD. Using multiphoton microscopy, Pittsburgh compound B (PIB) was imaged with sub-μm resolution in the brains of living transgenic mice during peripheral administration. PIB entered the brain quickly and labeled amyloid deposits within minutes. The nonspecific binding was cleared rapidly, whereas specific labeling was prolonged. WT mice showed rapid brain entry and clearance of PIB without any binding. These results demonstrate that the compound PIB has the properties required for a good amyloid-imaging agent in humans with or at risk for AD.

Development of a PET/SPECT agent for amyloid imaging in Alzheimer’s disease

In the search for a cure for Alzheimer’s disease (AD), efforts have been focused on preventing or reversing amyloid deposition in the brain. Efficacy evaluation of these antimyloid therapies would greatly benefit from development of a tool for the in vivo detection and quantitation of amyloid deposits in the brain. Toward this goal, we have developed a series of benzothiazole derivatives as amyloid-imaging agents for positron emission tomography (PET). To extend the potential of these amyloid-imaging agents for routine clinical studies, we also set out to develop iodinated benzothiazole derivatives that could be used as dual agents for either PET or the complementary single photon emission computed tomography (SPECT). Such dual agents would permit PET or SPECT studies using radiotracers with the same chemical identity but labeled with different radionuclides. This would facilitate the validation of clinical SPECT studies, based on quantitative PET studies. In this work we report the synthesis and biological evaluation of a potent, selective, and brain-permeable benzothiazole compound, 2-(3′-iodo-4′-methylaminophenyl)-6-hydroxy-benzothialzole, termed 6-OH-BTA-1-3′-I (4), which can be radiolabeled with either positron-emitting carbon-11 or single photon-emitting iodine-125/iodine-123. The synthesis and radiolabeling of [125I]4 or [11C]4 were achieved through direct iodination with sodium [125I]iodide in the presence of chloramine T or through radiomethylation with [11C]CH3I. In vitro amyloid binding assays indicated that [125I]4 bound to amyloid deposits in a saturable manner and exhibited affinities in the nanomolar concentration range. Binding studies of [125I]4 to postmortem human brain homogenates also showed preference of binding to frontal cortex in the AD homogenates relative to age-matched control homogenates or cerebellum from either AD or control. In vivo pharmacokinetic studies in normal mice following iv injection of [11C]4 indicated that the radioligand entered the brain readily at early time points and cleared from the brain rapidly at later time points with a 2- to 30-min ratio >3. These results suggest that the new radioiodinated benzothiazole ligand might be useful as a surrogate marker for the in vivo quantitation of mayloid deposition in human brain for use with either PET or SPECT.

Synthesis and evaluation of 2-(3′-iodo-4′-aminophenyl)-6-hydroxybenzothiazole for in vivo quantitation of amyloid deposits in Alzheimer's disease

A potent and brain permeable amyloid ligand has been identified as a lead compound capable of I-123/125-labelling for single photon emission computed tomography (SPECT) imaging. In this study, we report the synthesis and I-125-radiolabelling of Compound 6 and its in vitro and in vivo properties. Compound 6 [2-(3′-iodo-4′-aminophenyl)-6-hydroxybenzothiazole] bound to synthetic Aβ(1–40) fibrils in a saturable manner, exhibiting an affinity (Ki) of 11±1.1 nM in a competitive binding assay using a tritiated thioflavin T analog ([3H]BTA-1) as radioligand. [125I]6 binding to synthetic Aβ(1–40) fibrils fit a single-site model. [125I]6 exhibited several-fold higher binding to homogenates of frontal cortex from post-mortem Alzheimer’s disease brain relative to age-matched control brain homogenates. No difference in binding was observed in cerebellum. The ratio of radioactivity concentration between frontal cortex and cerebellum was 6-fold higher in AD brain homogenates than the age-matched control. [125I]6 also readily penetrated the blood-brain barrier in normal control mice with an average radioactivity concentration of 6.43 ± 0.62%ID/g detected in the whole brain at 2 min post i.v. injection. At 30 min, the radioactivity concentration decreased to 0.40 ± 0.05%ID/g, indicating good clearance in the absence of amyloid deposits in the brain.

Effects of lipophilicity on the affinity and nonspecific binding of iodinated benzothiazole derivatives.

A series of novel 2-aryl benzothiazole derivates substituted with iodine in different positions have been synthesized as amyloid-binding ligands. The affinity of these compounds for synthetic amyloid β (1–40) (Aβ[1–40]) fibrils was determined. Introduction of the iodo group in the position ortho to an amino group increased the binding affinity, whereas the iodination ortho to a hydroxyl group decreased the binding affinity. Selected compounds with high binding affinity and moderate lipophilicity (logP values, 1.65–3.90) were radiolabeled and evaluated in normal mice for brain uptake and clearance. Structure-activity relationship (SAR) studies showed a strong correlation between the lipophilicity of the iodinated compounds and the binding affinity as well as nonspecific binding. As the lipophilicity increased, the affinity for Aβ(1–40) fibrils improved; however, nonspecific binding in mouse brain reflected by low brain clearance also increased with increasing lipophilicity. These results provide important SAR information to guide the development of novel amyloid-binding agents and provide further insights into the molecular interaction between 2-aryl benzothiazole ligands and Aβ fibrils.