Andrew G. Horti

Fibroblast activation protein (FAP) is a validated PET imaging target for the tumor microenvironment. Currently, most 18F-labeled FAP PET agents use Al−F chemistry, with Al18F-FAPI-74 leading clinically, whereas C−18F tracers perform less effectively. Here, we developed (4-quinolinoyl)glycyl-2-cyanopyrrolidine (QCP)-based inhibitors incorporating a 6-fluoronicotinamide prosthetic group for stable C−18F labeling. Chelator incorporation improved hydrophilicity and enabled structurally matched radiometal- and 18F-labeled analogs from a common scaffold. The compounds exhibited high FAP inhibitory capacity (Ki = 9.9−56.7 pM) and favorable physicochemical properties (Log P7.4 = −3.1 ± 0.1). The lead tracer, [18F]FPyQCP, was prepared by a one-pot, two-step radiosynthesis in 50−80% yields, with >99% radiochemical purity and molar activity of 74−200 GBq/μmol. In U87 xenografts, [18F]FPyQCP achieved high tumor uptake (23.3 ± 2.1 %ID/g) and favorable tumor-to-blood ratios (12.0 ± 0.3) at 2 h. This modular labeling strategy establishes a versatile platform for next-generation FAP PET tracers and beyond.

Positron emission tomography is a useful tool for quantitative imaging of neuroinflammation in human subjects; however, more suitable radiotracers are soug

BACKGROUND: Microglia-mediated brain immune changes play a role in the pathogenesis of Parkinson’s disease (PD), but imaging microglia in living people with PD has relied on positron emission tomography (PET) ligands that lack specificity in labeling immune cells in the nervous system. We aimed to develop imaging of colony stimulating factor 1 receptor (CSF1R) as a microglial-sensitive marker of innate immunity. METHODS: IHC using a CSF1R antibody evaluated colocalization with Iba-1 in PD ( n = 4) and control ( n = 4) human brain samples. Autoradiography using a CSF1R tritiated ligand in human brain samples from individuals with PD ( n = 5) and in a control group ( n = 4) was performed to obtain B _max . PET imaging using a CSF1R radioligand was performed in 10 controls and 12 people with PD, and V _T was compared between groups and correlated with disease severity. RESULTS: IHC of CSF1R in human brain samples shows colocalization with Iba-1 and is significantly increased in brain samples from individuals with PD compared with individuals in a control group. Autoradiography revealed significantly increased CSF1R ligand binding in the inferior parietal cortex of patients with PD. [ ¹¹ C]CPPC PET showed higher binding in people with moderate PD compared with people in a control group and ligand binding correlated with more severe motor disability and poorer verbal fluency. CONCLUSION: This study underscores the significance of CSF1R imaging as a promising biomarker for brain immune function in Parkinson’s disease, which may be associated with cognitive and motor disease severity. FUNDING: PET imaging: the Michael J. Fox Foundation and the RMS Family Foundation. Radiotracer development: NIH (R01AG066464 and P41 EB024495). Postmortem brain tissues: NIH P30 AG066507 and BIOCARD study NIH U19 AG033655.

Background: Reactive microglia and recruited peripheral macrophages contribute to the pathogenesis of Alzheimer’s dementia (AD). Monocytes, macrophages and microglia all express the marker colony-stimulating factor 1 receptor (CSF1R). 4-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)-1H-pyrrole-2-carboxamide (1) is a high-affinity antagonist for CSF1R. We report the radiosynthesis of both [³H]1 and [¹¹C]1. The PET imaging properties of [¹¹C]1 in mice and baboon were investigated. [³H]1 was studied in B_max measurement in post-mortem autoradiography in the frontal cortex, inferior parietal cortex and hippocampus from donors diagnosed with AD and age-matched controls. In vitro binding affinity of 1 was measured commercially. Nor-methyl-1 precursor was radiolabeled with [¹¹C]iodomethane or [³H]iodomethane to produce [¹¹C]1 and [³H]1, respectively. Ex vivo brain biodistribution of [¹¹C]1 was compared in normal mice versus lipopolysaccharide-administered (LPS) murine model of neuroinflammation. Dynamic PET imaging was performed in a healthy male Papio anubis baboon. Post-mortem autoradiography with [³H]1 was performed in frozen sections using a standard saturation binding technique. Results: Compound 1 exhibits a high in vitro CSF1R binding affinity (0.59 nM). [¹¹C]1 was synthesized with high yield. [³H]1 was synthesized similarly (commercially). Biodistribution of [¹¹C]1 in healthy mice demonstrated moderate brain uptake. In LPS-treated mice the brain uptake of [¹¹C]1 was ~ 50% specific for CSF1R. PET/CT [¹¹C]1 study in baboon revealed low brain uptake (0.36 SUV) of [¹¹C]1. Autoradiography with [³H]1 gave significantly elevated B_max values in AD frontal cortex versus control (47.78 ± 26.80 fmol/mg vs. 12.80 ± 5.30 fmol/mg, respectively, P = 0.023) and elevated, but not significantly different binding in AD hippocampus grey matter and inferior parietal cortex (IPC) white matter. Conclusions: Compound 1 exhibits a high in vitro CSF1R binding affinity. [¹¹C]1 specifically labels CSF1R in the mouse neuroinflammation, but lacks the ability to efficiently cross the blood–brain barrier in baboon PET. [³H]1 specifically labels CSF1R in post-mortem human brain. The binding of [³H]1 is significantly higher in the post-mortem frontal cortex of AD versus control subjects.

Neuroinflammation through enhanced innate immunity is thought play a role in the pathogenesis of Parkinson’s disease (PD). Methods for monitoring neuroinflammation in living patients with PD are currently limited to positron emission tomography (PET) ligands that lack specificity in labeling immune cells in the nervous system. The colony stimulating factor 1 receptor (CSF1R) plays a crucial role in microglial function, an important cellular contributor to the nervous system’s innate immune response. Using immunologic methods, we show that CSF1R in human brain is colocalized with the microglial marker, ionized calcium binding adaptor molecule 1 (Iba1). In PD, CSF1R immunoreactivity is significantly increased in PD across multiple brain regions, with the largest differences in the midbrain versus controls. Autoradiography revealed significantly increased [ ³ H]JHU11761 binding in the inferior parietal cortex of PD patients. PET imaging demonstrated that higher [ ¹¹ C]CPPC binding in the striatum was associated with greater motor disability in PD. Furthermore, increased [ ¹¹ C]CPPC binding in various regions correlated with more severe motor disability and poorer verbal fluency. This study finds that CSF1R expression is elevated in PD and that [ ¹¹ C]CPPC-PET imaging of CSF1R is indicative of motor and cognitive impairments in the early stages of the disease. Moreover, the study underscores the significance of CSF1R as a promising biomarker for neuroinflammation in Parkinson’s disease, suggesting its potential use for non-invasive assessment of disease progression and severity, leading to earlier diagnosis and targeted interventions. Significance Statement: This study demonstrates that the Colony Stimulating Factor 1 Receptor (CSF1R) colocalizes with microglial markers in the human brain, and the research establishes elevated CSF1R expression in PD autopsy tissues. Employing [ ¹¹ C]CPPC-PET imaging, the study unveils a correlation between increased CSF1R binding and both motor disability and cognitive decline in PD patients. These findings highlight the potential of CSF1R as a novel biomarker for neuroinflammation in PD, offering a non-invasive means to assess disease progression and severity, ultimately contributing to earlier diagnosis and targeted interventions.

Purpose: Soluble epoxide hydrolase (sEH) is a promising candidate positron emission tomography (PET) imaging biomarker altered in various disorders, including vascular cognitive impairment (VCI), Alzheimer’s disease (AD), Parkinson’s disease (PD), stroke, and depression, known to regulate levels of epoxyeicosatrienoic acids (EETs) and play an important role in neurovascular coupling. [¹⁸F]FNDP, a PET radiotracer for imaging sEH, was evaluated through quantitative PET imaging in the baboon brain, radiometabolite analysis, and radiation dosimetry estimate. Methods: Baboon [¹⁸F]FNDP dynamic PET studies were performed at baseline and with blocking doses of the selective sEH inhibitor AR-9281 to evaluate sEH binding specificity. Radiometabolites of [¹⁸F]FNDP in mice and baboons were measured by high-performance liquid chromatography. Regional brain distribution volume (V_T) of [¹⁸F]FNDP was computed from PET using radiometabolite-corrected arterial input functions. Full body distribution of [¹⁸F]FNDP was studied in CD-1 mice, and the human effective dose was estimated using OLINDA/EXM software. Results: [¹⁸F]FNDP exhibited high and rapid brain uptake in baboons. AR-9281 blocked [¹⁸F]FNDP uptake dose-dependently with a baseline V_T of 10.9 ± 2.4 mL/mL and a high-dose blocking V_T of 1.0 ± 0.09 mL/mL, indicating substantial binding specificity (91.70 ± 1.74%). The V_ND was estimated as 0.865 ± 0.066 mL/mL. The estimated occupancy values of AR-9281 were 99.2 ± 1.1% for 1 mg/kg, 88.6 ± 1.3% for 0.1 mg/kg, and 33.8 ± 3.8% for 0.02 mg/kg. Murine biodistribution of [¹⁸F]FNDP enabled an effective dose estimate for humans (0.032 mSv/MBq). [¹⁸F]FNDP forms hydrophilic radiometabolites in murine and non-human primate plasma. However, only minute amounts of the radiometabolites entered the animal brain (< 2% in mice). Conclusions: [¹⁸F]FNDP is a highly sEH-specific radiotracer that is suitable for quantitative PET imaging in the baboon brain. [¹⁸F]FNDP holds promise for translation to human subjects.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in several cognitive and physiologic processes; its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer’s disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [¹²⁵I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(¹²⁵I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [¹⁸F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [¹²⁵I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [¹²⁵I]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in β2 nAChR gene-deficient mice was lower for [¹²⁵I]Iodo-ASEM (58% ± 2.7%) than [¹²⁵I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7β2 nAChR. [¹⁸F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [¹⁸F]ASEM binding. Our findings indicate that [¹²⁵I]Iodo-ASEM allows sensitive and selective imaging of α7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [¹⁸F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of α7 nAChR, comparable to previously published data.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated ¹⁸F positron emission tomography (PET) DREADD radiotracer, [¹⁸F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [¹⁸F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.