A Philosophy for CNS Radiotracer Design

Conspectus Decades after its discovery, positron emission tomography (PET) remains the premier tool for imaging neurochemistry in living humans. Technological improvements in radiolabeling methods, camera design, and image analysis have kept PET in the forefront. In addition, the use of PET imaging has expanded because researchers have developed new radiotracers that visualize receptors, transporters, enzymes, and other molecular targets within the human brain. However, of the thousands of proteins in the central nervous system (CNS), researchers have successfully imaged fewer than 40 human proteins. To address the critical need for new radiotracers, this Account expounds on the decisions, strategies, and pitfalls of CNS radiotracer development based on our current experience in this area. We discuss the five key components of radiotracer development for human imaging: choosing a biomedical question, selection of a biological target, design of the radiotracer chemical structure, evaluation of candidate radiotracers, and analysis of preclinical imaging. It is particularly important to analyze the market of scientists or companies who might use a new radiotracer and carefully select a relevant biomedical question(s) for that audience. In the selection of a specific biological target, we emphasize how target localization and identity can constrain this process and discuss the optimal target density and affinity ratios needed for binding-based radiotracers. In addition, we discuss various PET test–retest variability requirements for monitoring changes in density, occupancy, or functionality for new radiotracers. In the synthesis of new radiotracer structures, high-throughput, modular syntheses have proved valuable, and these processes provide compounds with sites for late-stage radioisotope installation. As a result, researchers can manage the time constraints associated with the limited half-lives of isotopes. In order to evaluate brain uptake, a number of methods are available to predict bioavailability, blood–brain barrier (BBB) permeability, and the associated issues of nonspecific binding and metabolic stability. To evaluate the synthesized chemical library, researchers need to consider high-throughput affinity assays, the analysis of specific binding, and the importance of fast binding kinetics. Finally, we describe how we initially assess preclinical radiotracer imaging, using brain uptake, specific binding, and preliminary kinetic analysis to identify promising radiotracers that may be useful for human brain imaging. Although we discuss these five design components separately and linearly in this Account, in practice we develop new PET-based radiotracers using these design components nonlinearly and iteratively to develop new compounds in the most efficient way possible

Strategy for Dual-Analyte Luciferin Imaging: <i>In Vivo</i> Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation

<i>In vivo</i> molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs <i>in situ</i> formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H₂O₂-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-d-Cys (IETDC), which releases d-cysteine in the presence of active caspase 8. Once released, HCBT and d-cysteine form firefly luciferin <i>in situ</i>, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H₂O₂ and caspase 8 activity. Using these probes, chemoselective imaging of either H₂O₂ or caspase 8 activity was performed <i>in vitro</i> and <i>in vivo</i>. Moreover, concomitant use of PCL-2 and IETDC <i>in vivo</i> establishes a concurrent increase in both H₂O₂ and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies

In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease.

Copper is a required metal nutrient for life, but global or local alterations in its homeostasis are linked to diseases spanning genetic and metabolic disorders to cancer and neurodegeneration. Technologies that enable longitudinal in vivo monitoring of dynamic copper pools can help meet the need to study the complex interplay between copper status, health, and disease in the same living organism over time. Here, we present the synthesis, characterization, and in vivo imaging applications of Copper-Caged Luciferin-1 (CCL-1), a bioluminescent reporter for tissue-specific copper visualization in living animals. CCL-1 uses a selective copper(I)-dependent oxidative cleavage reaction to release d-luciferin for subsequent bioluminescent reaction with firefly luciferase. The probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of nonalcoholic fatty liver disease reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. The data connect copper dysregulation to metabolic liver disease and provide a starting point for expanding the toolbox of reactivity-based chemical reporters for cell- and tissue-specific in vivo imaging

Positron Emission Tomography Assessment of the Intranasal Delivery Route for Orexin A

Intranasal drug delivery is a noninvasive drug delivery route that can enhance systemic delivery of therapeutics with poor oral bioavailability by exploiting the rich microvasculature within the nasal cavity. The intranasal delivery route has also been targeted as a method for improved brain uptake of neurotherapeutics, with a goal of harnessing putative, direct nose-to-brain pathways. Studies in rodents, nonhuman primates, and humans have pointed to the efficacy of intranasally delivered neurotherapeutics, while radiolabeling studies have analyzed brain uptake following intranasal administration. In the present study, we employed carbon-11 radioactive methylation to assess the pharmacokinetic mechanism of intranasal delivery of Orexin A, a native neuropeptide and prospective antinarcoleptic drug that binds the orexin receptor 1. Using physicochemical and pharmacological analysis, we identified the methylation sites and confirmed the structure and function of methylated Orexin A (CH₃-Orexin A) prior to monitoring its brain uptake following intranasal administration in rodent and nonhuman primate. Through positron emission tomography (PET) imaging of [¹¹C]­CH₃-Orexin A, we determined that the brain exposure to Orexin A is poor after intranasal administration. Additional ex vivo analysis of brain uptake using [¹²⁵I]­Orexin A indicated intranasal administration of Orexin A affords similar brain uptake when compared to intravenous administration across most brain regions, with possible increased brain uptake localized to the olfactory bulbs

In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease

Copper is a required metal nutrient for life, but global or local alterations in its homeostasis are linked to diseases spanning genetic and metabolic disorders to cancer and neurodegeneration. Technologies that enable longitudinal in vivo monitoring of dynamic copper pools can help meet the need to study the complex interplay between copper status, health, and disease in the same living organism over time. Here, we present the synthesis, characterization, and in vivo imaging applications of Copper-Caged Luciferin-1 (CCL-1), a bioluminescent reporter for tissue-specific copper visualization in living animals. CCL-1 uses a selective copper(I)-dependent oxidative cleavage reaction to release d-luciferin for subsequent bioluminescent reaction with firefly luciferase. The probe can detect physiological changes in labile Cu(+) levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of nonalcoholic fatty liver disease reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. The data connect copper dysregulation to metabolic liver disease and provide a starting point for expanding the toolbox of reactivity-based chemical reporters for cell- and tissue-specific in vivo imaging

Development of a Fluorinated Class‑I HDAC Radiotracer Reveals Key Chemical Determinants of Brain Penetrance

Despite major efforts, our knowledge about many brain diseases remains remarkably limited. Epigenetic dysregulation has been one of the few leads toward identifying the causes and potential treatments of psychiatric disease over the past decade. A new positron emission tomography radiotracer, [¹¹C]­Martinostat, has enabled the study of histone deacetylase in living human subjects. A unique property of [¹¹C]­Martinostat is its profound brain penetrance, a feature that is challenging to engineer intentionally. In order to understand determining factors for the high brain-uptake of Martinostat, a series of compounds was evaluated in rodents and nonhuman primates. The study revealed the major structural contributors to brain uptake, as well as a more clinically relevant fluorinated HDAC radiotracer with comparable behavior to Martinostat, yet longer half-life

Nasal neuron PET imaging quantifies neuron generation and degeneration

Olfactory dysfunction is broadly associated with neurodevelopmental and neurodegenerative diseases and predicts increased mortality rates in healthy individuals. Conventional measurements of olfactory health assess odor processing pathways within the brain and provide a limited understanding of primary odor detection. Quantification of the olfactory sensory neurons (OSNs), which detect odors within the nasal cavity, would provide insight into the etiology of olfactory dysfunction associated with disease and mortality. Notably, OSNs are continually replenished by adult neurogenesis in mammals, including humans, so OSN measurements are primed to provide specialized insights into neurological disease. Here, we have evaluated a PET radiotracer, [11C]GV1-57, that specifically binds mature OSNs and quantifies the mature OSN population in vivo. [11C]GV1-57 monitored native OSN population dynamics in rodents, detecting OSN generation during postnatal development and aging-associated neurodegeneration. [11C]GV1-57 additionally measured rates of neuron regeneration after acute injury and early-stage OSN deficits in a rodent tauopathy model of neurodegenerative disease. Preliminary assessment in nonhuman primates suggested maintained uptake and saturable binding of [18F]GV1-57 in primate nasal epithelium, supporting its translational potential. Future applications for GV1-57 include monitoring additional diseases or conditions associated with olfactory dysregulation, including cognitive decline, as well as monitoring effects of neuroregenerative or neuroprotective therapeutics