Evan Gallagher

Background/Objectives: Quantitative PET imaging of μ-opioid receptor (MOR) availability and pharmacologic engagement can advance in vivo studies of opioid receptor biology. We previously evaluated a series of ¹⁸F-labeled carfentanil analogs and prioritized the regioisomer [¹⁸F]fluorocarfentanil ([¹⁸F]FCFN) based on favorable brain kinetics, regional distribution, and naloxone-sensitive binding. Here, we provide proof-of-concept evaluation of [¹⁸F]FCFN in rhesus macaques to assess its potential for quantitative, pharmacologically responsive MOR PET. Methods: [¹⁸F]FCFN was produced by automated radiosynthesis. Dynamic [¹⁸F]FCFN PET scans were performed in two rhesus macaques under baseline conditions, after low- (0.005 mg/kg) and high-dose (0.2 mg/kg) naloxone pretreatment, and during within-scan intravenous or intranasal naloxone challenge. For arterial input scans, two-tissue compartment modeling with metabolite-corrected arterial input functions was used to estimate total distribution volume (V_T). Nondisplaceable binding potential (BP_ND) was estimated using multilinear reference tissue model 2 with the cerebellum as the candidate reference region. Results: Automated radiosynthesis produced [¹⁸F]FCFN in high radiochemical yield and molar activity, enabling low-mass PET injections (0.09–0.32 μg). [¹⁸F]FCFN showed rapid brain uptake and MOR-consistent regional distribution, with lowest uptake in the cerebellum. Regional BP_ND correlated positively with V_T at baseline (R² = 0.983, p < 0.0001) and after high-dose blockade (R² = 0.522, p < 0.004). Naloxone pretreatment reduced regional BP_ND in a dose-dependent manner. Within-scan intravenous and intranasal naloxone challenges also produced marked reductions in BP_ND. Conclusions: [¹⁸F]FCFN enables quantitative, pharmacologically sensitive MOR PET imaging in nonhuman primates at low injected masses. Concordant plasma-input and reference-tissue measures, dose-dependent blockade, and proof-of-concept within-scan displacement support its use for measuring MOR availability and dynamic antagonist engagement across administration routes.

Oxidative stress, defined as the excessive production of reactive oxygen species (ROS), is a crucial factor in the pathogenesis of various neurodegenerative diseases, including the 4-repeat (4R) tauopathies. Collectively, the 4R tauopathies are characterized by the progressive aggregation of tau protein isoforms with four microtubule-binding domains in and around brain cells. The cyclical relationship between oxidative stress and 4R tau aggregation suggests that a means of imaging ROS noninvasively could be a valuable tool for the study and treatment of 4R tauopathy in both humans and animal models. To demonstrate the potential of the ROS-sensitive positron emission tomography (PET) radiotracer [¹⁸F]ROStrace as a means of filling this methodological gap, we performed [¹⁸F]ROStrace PET imaging on PS19 mice, which exhibit 4R tau aggregation similar to that seen in human 4R tauopathy. Significant increases in [¹⁸F]ROStrace signal became detectable in the hippocampus of 6–11-month-old (mo) PS19 animals and spread to the brainstem, midbrain, and thalamus of 11+ mo animals. Additionally, older PS19 mice displayed higher whole-brain average [¹⁸F]ROStrace signal compared to age-matched controls (p = 0.042), and tau pathology consistently colocalized with multiple fluorescent indicators of oxidative stress in PS19 brain samples. These results provide novel evidence that 4R tau aggregation is associated with increased oxidative stress in PS19 mouse brain and advance [¹⁸F]ROStrace as a noninvasive technology for the detection of oxidative stress in neurodegenerative diseases involving tau pathology.

The synucleinopathies are a diverse group of neurodegenerative disorders characterized by the accumulation of aggregated alpha-synuclein (aSyn) in vulnerable populations of brain cells. Oxidative stress is both a cause and a consequence of aSyn aggregation in the synucleinopathies; however, noninvasive methods for detecting oxidative stress in living animals have proven elusive. In this study, we used the reactive oxygen species (ROS)-sensitive positron emission tomography (PET) radiotracer [¹⁸F]ROStrace to detect increases in oxidative stress in the widely-used A53T mouse model of synucleinopathy. A53T-specific elevations in [¹⁸F]ROStrace signal emerged at a relatively early age (6–8 months) and became more widespread within the brain over time, a pattern which paralleled the progressive development of aSyn pathology and oxidative damage in A53T brain tissue. Systemic administration of lipopolysaccharide (LPS) also caused rapid and long-lasting elevations in [¹⁸F]ROStrace signal in A53T mice, suggesting that chronic, aSyn-associated oxidative stress may render these animals more vulnerable to further inflammatory insult. Collectively, these results provide novel evidence that oxidative stress is an early and chronic process during the development of synucleinopathy and suggest that PET imaging with [¹⁸F]ROStrace holds promise as a means of detecting aSyn-associated oxidative stress noninvasively.

This study assessed whether the newly developed PET radioligands ¹¹C-PS13 and ¹¹C-MC1 could image constitutive levels of cyclooxygenase-1 (COX-1) and COX-2, respectively, in rhesus monkeys. Methods: After intravenous injection of either radioligand, 24 whole-body PET scans were performed. To measure enzyme-specific uptake, scans of the two radioligands were also performed after administration of a non-radioactive drug preferential for either COX-1 or COX-2. Concurrent venous samples were obtained to measure parent radioligand concentrations. Standardized uptake values were calculated from 10-90 minutes. Results: ¹¹C-PS13 showed specific uptake in most organs including spleen, gastrointestinal tract, kidneys, and brain, which was blocked by COX-1, but not COX-2, preferential inhibitors. Specific uptake of ¹¹C-MC1 was not observed in any organ except the ovaries and possibly kidneys. Conclusion: The findings suggest that ¹¹C-PS13 has adequate signal in monkeys to justify its extension to human subjects. In contrast, ¹¹C-MC1 is unlikely to show significant signal in healthy humans, though it may be able to do so in inflammatory conditions.

Translocator protein 18 kDa (TSPO) has been widely imaged as a marker of neuroinflammation using several radioligands, including [¹¹C]PBR28. In order to study the effects of age, sex, and obesity on TSPO binding and to determine whether this binding can be accurately assessed using fewer radio high-performance liquid chromatography (radio-HPLC) measurements of arterial blood samples, we created a database of 48 healthy subjects who had undergone [¹¹C]PBR28 scans (23 high-affinity binders (HABs) and 25 mixed-affinity binders (MABs), 20 F/28 M, age: 40.6 ± 16.8 years). After analysis by Logan plot using 23 metabolite-corrected arterial samples, total distribution volume ( V_T) was found to be 1.2-fold higher in HABs across all brain regions. Additionally, the polymorphism plot estimated nondisplaceable uptake ( V_ND) as 1.40 mL · cm⁻³, which generated a specific-to-nondisplaceable ratio ( BP_ND) of 1.6 ± 0.6 in HABs and 1.1 ± 0.6 in MABs. V_T increased significantly with age in nearly all regions and was well estimated with radio-HPLC measurements from six arterial samples. However, V_T did not correlate with body mass index and was not affected by sex. These results underscore which patient characteristics should be accounted for during [¹¹C]PBR28 studies and suggest ways to perform such studies more easily and with fewer blood samples.