Manik L. Debnath

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.

Post-mortem correlates of in vivo PiB-PET amyloid imaging in a typical case of Alzheimer's disease

The positron emission tomography (PET) radiotracer Pittsburgh Compound-B (PiB) binds with high affinity to β-pleated sheet aggregates of the amyloid-β (Aβ) peptide in vitro. The in vivo retention of PiB in brains of people with Alzheimers disease shows a regional distribution that is very similar to distribution of Aβ deposits observed post-mortem. However, the basis for regional variations in PiB binding in vivo, and the extent to which it binds to different types of Aβ-containing plaques and tau-containing neurofibrillary tangles (NFT), has not been thoroughly investigated. The present study examined 28 clinically diagnosed and autopsy-confirmed Alzheimers disease subjects, including one Alzheimers disease subject who had undergone PiB-PET imaging 10 months prior to death, to evaluate region- and substrate-specific binding of the highly fluorescent PiB derivative 6-CN-PiB. These data were then correlated with region-matched Aβ plaque load and peptide levels, [3H]PiB binding in vitro, and in vivo PET retention levels. We found that in Alzheimers disease brain tissue sections, the preponderance of 6-CN-PiB binding is in plaques immunoreactive to either Aβ42 or Aβ40, and to vascular Aβ deposits. 6-CN-PiB labelling was most robust in compact/cored plaques in the prefrontal and temporal cortices. While diffuse plaques, including those in caudate nucleus and presubiculum, were less prominently labelled, amorphous Aβ plaques in the cerebellum were not detectable with 6-CN-PiB. Only a small subset of NFT were 6-CN-PiB positive; these resembled extracellular ‘ghost’ NFT. In Alzheimers disease brain tissue homogenates, there was a direct correlation between [3H]PiB binding and insoluble Aβ peptide levels. In the Alzheimers disease subject who underwent PiB-PET prior to death, in vivo PiB retention levels correlated directly with region-matched post-mortem measures of [3H]PiB binding, insoluble Aβ peptide levels, 6-CN-PiB- and Aβ plaque load, but not with measures of NFT. These results demonstrate, in a typical Alzheimers disease brain, that PiB binding is highly selective for insoluble (fibrillar) Aβ deposits, and not for neurofibrillary pathology. The strong direct correlation of in vivo PiB retention with region-matched quantitative analyses of Aβ plaques in the same subject supports the validity of PiB-PET imaging as a method for in vivo evaluation of Aβ plaque burden.

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.

Validating novel tau positron emission tomography tracer [F-18]-AV-1451 (T807) on postmortem brain tissue

To examine region‐ and substrate‐specific autoradiographic and in vitro binding patterns of positron emission tomography tracer [F‐18]‐AV‐1451 (previously known as T807), tailored to allow in vivo detection of paired helical filament‐tau–containing lesions, and to determine whether there is off‐target binding to other amyloid/non‐amyloid proteins.

Development of small molecule probes for the beta-amyloid protein of alzheimer's disease

This study describes the synthesis and in vitro testing of small molecule probes that may eventually prove useful as markers of amyloid deposition in living patients. The prototype agent, Chrysamine G (CG), is a derivative of Congo red. CG binds synthetic beta-amyloid well in vitro, as does a fluorinated derivative. The mechanism of binding appears to be the same as Congo red--through a bidentate attachment spanning several amyloid peptide chains. CG is much more lipophilic than Congo red and crosses the blood-brain barrier in normal mice, achieving a brain/blood ratio over 10/1. There was no acute toxicity in mice at doses 10 times those used in the distribution studies. CG appears to be a relatively high affinity probe for beta-amyloid that appears to have low toxicity and can cross the blood-brain barrier. These characteristics are promising for development of in vivo amyloid probes similar to CG.

Chrysamine-G binding to Alzheimer and control brain : autopsy study of a new amyloid probe

Chrysamine-G (CG) is a carboxylic acid analogue of Congo red, a histologic dye which stains amyloid. CG binds to the beta-amyloid protein of Alzheimers disease (AD) in vitro and partitions into the brain of normal mice. In this study, we demonstrate increased binding of [14C]CG to homogenates of several regions of AD brain as compared to control. The total binding of CG to AD brain was approximately two- to three-fold that of control brain. The cerebellum could be used as an internal standard for each brain as CG binding to cerebellum did not differ between AD and control. The binding of [14C]CG correlated with numbers of senile plaques and neurofibrillary tangles. In addition, CG could be used to stain cerebrovascular amyloid in tissue sections. These results suggest that CG may prove useful as an in vivo probe of amyloid deposition in AD.

Chrysamine-G, a lipophilic analogue of congo red, inhibits Aβ-induced toxicity in PC12 cells

Increasing evidence suggests that deposition of amyloid-beta (A beta) peptide leads to neurodegeneration in Alzheimers disease. Congo red, a histologic dye that binds to amyloid has previously been shown to diminish the toxic effects of A beta in cell culture. Since Congo red is too highly charged to enter the brain in significant quantities, a lipophilic derivative, Chrysamine-G, was tested for the ability to attenuate A beta[25-35]-induced toxicity in PC12 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chrysamine-G showed a concentration-dependent inhibition of A beta[25-35]-induced toxicity. This protective effect became significant at 0.2 microM, a concentration very close to the Ki for Chrysamine-G binding to synthetic A beta (0.37 microM). A decarboxy derivative of Chrysamine-G, which does not bind to A beta, also did not protect against A beta-induced toxicity. The protective effects of Chrysamine-G may relate to its ability to bind directly to A beta and may involve other post-binding effects as well.

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.