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Senile plaques (SPs) and neurofibrillary tangles (NFTs) are hallmark pathologies accompanying the neurodegeneration involved in Alzheimer's disease (AD), for which beta-amyloid (Abeta) peptide is a major constituent of SPs. Our laboratories previously developed the hydrophobic, fluorescent molecular-imaging probe 2-(1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl)ethylidene)malononitrile ([(18)F]FDDNP), which crosses the blood-brain barrier and determines the localization and load of SPs and NFTs in vivo in AD patients. In this report, we used fluorimetric and radioactive binding assays to determine the binding affinities of FDDNP and its analog, 1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]naphthalen-2-yl)ethanone ([(18)F]FENE), to synthetic fibrils of Abeta(1-40). FDDNP and FENE both appeared to bind to two kinetically distinguishable binding sites on Abeta(1-40) fibrils. Fluorescence titrations yielded apparent K(d) values of 0.12 and 0.16 nm for high-affinity binding sites for FDDNP and FENE, respectively, and apparent K(d) values of 1.86 and 71.2 nm for the low-affinity binding sites. The traditional radioactive binding assays also produced apparent K(d) values in the low nanomolar range. The presence of two kinetically distinguishable binding sites for FDDNP and FENE suggests multiple binding sites for SPs and identifies the parameters that allow for the structural optimization of this family of probes for in vivo use. The high-affinity binding of the probes to multiple binding sites on fibrils are consistent with results obtained with digital autoradiography, immunohistochemistry, and confocal fluorescence microscopy using human brain specimens of AD patients.

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This educational review highlights the processes, opportunities, and challenges encountered in the discovery and development of imaging agents, mainly positron emission tomography and single-photon emission computed tomography tracers. While the development of imaging agents parallels the drug development process, unique criteria are needed to identify opportunities for new agents. Imaging agent development has the flexibility to pursue functional or nonfunctional targets as long as they play a role in the specific disease or mechanism of interest and meet imageability requirements. However, their innovation is tempered by relatively small markets for diagnostic imaging agents, intellectual property challenges, radiolabeling constraints, and adequate target concentrations for imaging. At the same time, preclinical imaging is becoming a key translational tool for proof of mechanism and concept studies. Pharmaceutical and imaging industries face a common bottleneck in the form of the limited number of trials one company can possibly perform. However, microdosing and theranostics are evidence that partnerships between pharmaceutical and imaging companies can accelerate clinical translation of tracers and therapeutic interventions. This manuscript will comment on these aspects to provide an educational review of the discovery and development processes for imaging agents.

Cerebral neurofibrillary tangles (NFTs) accumulate in a predictable sequence decades before the clinical symptoms of Alzheimer's disease emerge, and the degree of tangle degeneration correlates with the severity of cognitive impairment. A valid in vivo marker of tangle burden, therefore, would be useful for presymptomatic and symptomatic disease detection and treatment monitoring. Recent advances using positron emission tomography (PET) indicate the feasibility of in vivo imaging that provides a combined signal of both neurofibrillary tangles and senile plaques. Such results are encouraging that a tangle-specific marker will be found; however, several methodological issues first need to be addressed, including scanner spatial resolution in the relatively small brain regions where tangles accumulate. NFT-specific imaging probes will need to be lipophilic in order to cross the blood-brain barrier and neuronal membranes and have a high binding affinity to NFTs with minimal nonspecific binding, which would result in a high signal-to-background ratio in PET images.

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The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t -butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM K i ) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM K i ). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel.

Innovation in basic and applied science has brought radiotracers to fruition as diagnostics. Non-invasive, longitudinal, and quantifiable molecular imaging is the key to diagnosing and monitoring numerous illnesses, with more to come from characterization of the clinical relevance of findings from genomics research. Radiotracers enable real-time in vivo studies of the effects of drug candidates on receptors, pathways, pharmacodynamics, and clinically relevant endpoints, thereby providing both early detection of pathophysiology to enable early intervention, and then monitoring of treatment responses to enable individualization of treatment regimens. We review developments which have translated imaging from 'bench to bedside', or 'biomarkers to diagnostics'. Notable developments include (1) synthesis methods for rapid 11 C labeling of biomolecules to high specific radioactivity; (2) ligand-binding assays for screening molecular imaging agents rather than drugs; (3) in vivo imaging of radiotracers in animals; (4) discovering the imaging advantages of 99m Tc, 11 C, and 18 F; (5) co-registration and automated quantitative assessment of high spatial resolution CT and MR images with molecular images from PET for longitudinal studies of treatment effect.