Synthesis and Evaluation of [¹⁸F]RAGER: A First Generation Small-Molecule PET Radioligand Targeting the Receptor for Advanced Glycation Endproducts

The receptor for advanced glycation endproducts (RAGE) is a 35 kDa transmembrane receptor that belongs to the immunoglobulin superfamily of cell surface molecules. Its role in Alzheimer’s disease (AD) is complex, but it is thought to mediate influx of circulating amyloid-β into the brain as well as amplify Aβ-induced pathogenic responses. RAGE is therefore of considerable interest as both a diagnostic and a therapeutic target in AD. Herein we report the synthesis and preliminary preclinical evaluation of [¹⁸F]­RAGER, the first small molecule PET radiotracer for RAGE (<i>K</i>_d = 15 nM). Docking studies proposed a likely binding interaction between RAGE and RAGER, [¹⁸F]­RAGER autoradiography showed colocalization with RAGE identified by immunohistochemistry in AD brain samples, and [¹⁸F]­RAGER microPET confirmed CNS penetration and increased uptake in areas of the brain known to express RAGE. This first generation radiotracer represents initial proof-of-concept and a promising first step toward quantifying CNS RAGE activity using PET. However, there were high levels of nonspecific [¹⁸F]­RAGER binding <i>in vitro</i>, likely due to its high log <i>P</i> (experimental log <i>P</i> = 3.5), and rapid metabolism of [¹⁸F]­RAGER in rat liver microsome studies. Therefore, development of second generation ligands with improved imaging properties would be advantageous prior to anticipated translation into clinical PET imaging studies

Synthesis of Diverse ¹¹C‑Labeled PET Radiotracers via Direct Incorporation of [¹¹C]CO₂

Three new positron emission tomography (PET) radiotracers of interest to our functional neuroimaging and translational oncology programs have been prepared through new developments in [¹¹C]­CO₂ fixation chemistry. [¹¹C]­QZ (glutaminyl cyclase) was prepared via a tandem trapping of [¹¹C]­CO₂/intramolecular cyclization; [¹¹C]­tideglusib (glycogen synthase kinase-3) was synthesized through a tandem trapping of [¹¹C]­CO₂ followed by an intermolecular cycloaddition between a [¹¹C]­isocyanate and an isothiocyanate to form the 1,2,4-thiadiazolidine-3,5-dione core; [¹¹C]­ibrutinib (Bruton’s tyrosine kinase) was synthesized through a HATU peptide coupling of an amino precursor with [¹¹C]­acrylic acid (generated from [¹¹C]­CO₂ fixation with vinylmagnesium bromide). All radiochemical syntheses are fully automated on commercial radiochemical synthesis modules and provide radiotracers in 1–5% radiochemical yield (noncorrected, based upon [¹¹C]­CO₂). All three radiotracers have advanced to rodent imaging studies and preliminary PET imaging results are also reported

Targeting Metal-Aβ Aggregates with Bifunctional Radioligand [¹¹C]L2‑b and a Fluorine-18 Analogue [¹⁸F]FL2‑b

Interest in quantifying metal-Aβ species <i>in vivo</i> led to the synthesis and evaluation of [¹¹C]­L2-b and [¹⁸F]­FL2-b as radiopharmaceuticals for studying the metallobiology of Alzheimer’s disease (AD) using positron emission tomography (PET) imaging. [¹¹C]­L2-b was synthesized in 3.6% radiochemical yield (nondecay corrected, <i>n</i> = 3), >95% radiochemical purity, from the corresponding desmethyl precursor. [¹⁸F]­FL2-b was synthesized in 1.0% radiochemical yield (nondecay corrected, <i>n</i> = 3), >99% radiochemical purity, from a 6-chloro pyridine precursor. Autoradiography experiments with AD positive and healthy control brain samples were used to determine the specificity of binding for the radioligands compared to [¹¹C]­PiB, a known imaging agent for β-amyloid (Aβ) aggregates. The <i>K</i>_d for [¹¹C]­L2-b and [¹⁸F]­FL2-b were found to be 3.5 and 9.4 nM, respectively, from those tissue studies. Displacement studies of [¹¹C]­L2-b and [¹⁸F]­FL2-b with PiB and AV-45 determined that L2-b binds to Aβ aggregates differently from known radiopharmaceuticals. Finally, brain uptake of [¹¹C]­L2-b was examined through microPET imaging in healthy rhesus macaque, which revealed a maximum uptake at 2.5 min (peak SUV = 2.0) followed by rapid egress (<i>n</i> = 2)

Investigation of Proposed Activity of Clarithromycin at GABA_A Receptors Using [¹¹C]Flumazenil PET

Clarithromycin is a potential treatment for hypersomnia acting through proposed negative allosteric modulation of GABA_A receptors. We were interested whether this therapeutic benefit might extend to Parkinson’s disease (PD) patients because GABAergic neurotransmission is implicated in postural control. Prior to initiating clinical studies in PD patients, we wished to better understand clarithromycin’s mechanism of action. In this work we investigated whether the proposed activity of clarithromycin at the GABA_A receptor is associated with the benzodiazepine binding site using <i>in vivo</i> [¹¹C]­flumazenil positron emission tomography (PET) in primates and <i>ex vivo</i> [³H]­flumazenil autoradiography in rat brain. While the studies demonstrate that clarithromycin does not change the <i>K</i>_d of FMZ, nor does it competitively displace FMZ, there is preliminary evidence from the primate PET imaging studies that clarithromycin delays dissociation and washout of flumazenil from the primate brain in a dose-dependent fashion. These findings would be consistent with the proposed GABA_A allosteric modulator function of clarithromycin. While the results are only preliminary, further investigation of the interaction of clarithromycin with GABA receptors and/or GABAergic medications is warranted, and therapeutic applications of clarithromycin alone or in combination with flumazenil, to treat hyper-GABAergic status in PD at minimally effective doses, should also be pursued

Evaluation of [¹¹C]<i>N</i>‑Methyl Lansoprazole as a Radiopharmaceutical for PET Imaging of Tau Neurofibrillary Tangles

[¹¹C]<i>N</i>-Methyl lansoprazole ([¹¹C]­NML, <b>3</b>) was synthesized and evaluated as a radiopharmaceutical for quantifying tau neurofibrillary tangle (NFT) burden using positron emission tomography (PET) imaging. [¹¹C]­NML was synthesized from commercially available lansoprazole in 4.6% radiochemical yield (noncorrected RCY, based upon [¹¹C]­MeI), 99% radiochemical purity, and 16095 Ci/mmol specific activity (<i>n</i> = 5). Log <i>P</i> was determined to be 2.18. A lack of brain uptake in rodent microPET imaging revealed [¹¹C]­NML to be a substrate for the rodent permeability-glycoprotein 1 (PGP) transporter, but this could be overcome by pretreating with cyclosporin A to block the PGP. Contrastingly, [¹¹C]­NML was not found to be a substrate for the primate PGP, and microPET imaging in rhesus revealed [¹¹C]­NML uptake in the healthy primate brain of ∼1600 nCi/cc maximum at 3 min followed by rapid egress to 500 nCi/cc. Comparative autoradiography between wild-type rats and transgenic rats expressing human tau (hTau +/+) revealed 12% higher uptake of [¹¹C]­NML in the cortex of brains expressing human tau. Further autoradiography with tau positive brain samples from progressive supranuclear palsy (PSP) patients revealed colocalization of [¹¹C]­NML with tau NFTs identified using modified Bielschowsky staining. Finally, saturation binding experiments with heparin-induced tau confirmed <i>K</i>_d and Bmax values of [¹¹C]­NML as 700 pM and 0.214 fmol/μg, respectively

High Affinity Radiopharmaceuticals Based Upon Lansoprazole for PET Imaging of Aggregated Tau in Alzheimer’s Disease and Progressive Supranuclear Palsy: Synthesis, Preclinical Evaluation, and Lead Selection

Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [¹⁸F]­lansoprazole, [¹¹C]<i>N</i>-methyl lansoprazole, and [¹⁸F]<i>N</i>-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [¹⁸F]<i>N</i>-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials

Identification of [¹⁸F]TRACK, a Fluorine-18-Labeled Tropomyosin Receptor Kinase (Trk) Inhibitor for PET Imaging

Changes in expression and dysfunctional signaling of TrkA/B/C receptors and oncogenic Trk fusion proteins are found in neurological diseases and cancers. Here, we describe the development of a first ¹⁸F-labeled optimized lead suitable for in vivo imaging of Trk, [¹⁸F]­TRACK, which is radiosynthesized with ease from a nonactivated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads while displaying high on-target affinity and human kinome selectivity

A Kinome-Wide Selective Radiolabeled TrkB/C Inhibitor for in Vitro and in Vivo Neuroimaging: Synthesis, Preclinical Evaluation, and First-in-Human

The proto-oncogenes <i>NTRK1/2/3</i> encode the tropomyosin receptor kinases TrkA/B/C which play pivotal roles in neurobiology and cancer. We describe herein the discovery of [¹¹C]-(<i>R</i>)-<b>3</b> ([¹¹C]-(<i>R</i>)-IPMICF16), a first-in-class positron emission tomography (PET) TrkB/C-targeting radiolabeled kinase inhibitor lead. Relying on extensive human kinome vetting, we show that (<i>R</i>)-<b>3</b> is the most potent and most selective TrkB/C inhibitor characterized to date. It is demonstrated that [¹¹C]-(<i>R</i>)-<b>3</b> readily crosses the blood–brain barrier (BBB) in rodents and selectively binds to TrkB/C receptors in vivo, as evidenced by entrectinib blocking studies. Substantial TrkB/C-specific binding in human brain tissue is observed in vitro, with specific reduction in the hippocampus of Alzheimer’s disease (AD) versus healthy brains. We additionally provide preliminary translational data regarding the brain disposition of [¹¹C]-(<i>R</i>)-<b>3</b> in primates including first-in-human assessment. These results illustrate for the first time the use of a kinome-wide selective radioactive chemical probe for endogenous kinase PET neuroimaging in human