Because of its excellent ratio of specific to nondisplaceable uptake, the radioligand C-11-ER176 can successfully image 18-kDa translocator protein (TSPO), a biomarker of inflammation, in the human brain and accurately quantify target density in homozygous low-affinity binders. Our laboratory sought to develop an F-18-labeled TSPO PET radioligand based on ER176 with the potential for broader distribution. This study used generic C-11 labeling and in vivo performance in the monkey brain to select the most promising among 6 fluorine-containing analogs of ER176 for subsequent labeling with longer-lived F-18. Methods: Six fluorine-containing analogs of ER176-3 fluoro and 3 trifluoromethyl isomers-were synthesized and labeled by C-11 methylation at the secondary amide group of the respective N-desmethyl precursor. PET imaging of the monkey brain was performed at baseline and after blockade by N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide (PK11195). Uptake was quantified using radiometabolite-corrected arterial input function. The 6 candidate radioligands were ranked for performance on the basis of 2 in vivo criteria: the ratio of specific to nondisplaceable uptake (i.e., nondisplaceable binding potential [BPND]) and the time stability of total distribution volume (V-T), an indirect measure of lack of radiometabolite accumulation in the brain. Results: Total TSPO binding was quantified as V-T corrected for plasma free fraction (V-T/f(P)) using Logan graphical analysis for all 6 radioligands. V-T/f(P) was generally high at baseline (222 +/- 178 mL.cm(-3)) and decreased by 70%-90% after preblocking with PK11195. BPND calculated using the Lassen plot was 9.6 +/- 3.8; the o-fluoro radioligand exhibited the highest BPND (12.1), followed by the m-trifluoromethyl (11.7) and m-fluoro (8.1) radioligands. For all 6 radioligands, V-T reached 90% of the terminal 120-min values by 70 min and remained relatively stable thereafter, with excellent identifiability (SEs < 5%), suggesting that no significant radiometabolites accumulated in the brain. Conclusion: All 6 radioligands had good BPND and good time stability of V-T Among them, the o-fluoro, m-trifluoromethyl, and m-fluoro compounds were the 3 best candidates for development as radioligands with an F-18 label.

Translocator protein 18 kDa (TSPO) is a biomarker of neuroinflammation. [11C]ER176 robustly quantifies TSPO in the human brain with positron emission tomography (PET), irrespective of subject genotype. We aimed to develop an ER176 analog with potential for labeling with longer-lived fluorine-18 (t1/2 = 109.8 min). New fluoro and trifluoromethyl analogs of ER176 were prepared through a concise synthetic strategy. These ligands showed high TSPO affinity and low human genotype sensitivity. Each ligand was initially labeled by a generic 11C-methylation procedure, thereby enabling speedy screening in mice. Each radioligand was rapidly taken up and well retained in the mouse brain at baseline after intravenous injection. Preblocking of TSPO showed that high proportions of brain uptake were specifically bound to TSPO at baseline. Overall, the 3-fluoro analog of [11C]ER176 ([11C]3b) displayed the most promising imaging properties. Therefore, a method was developed to label 3b with [18F]fluoride ion. [18F]3b gave similarly promising PET imaging results and deserves evaluation in higher species.

Cyclooxygenase-1 (COX-1) and its isozyme COX-2 are key enzymes in the syntheses of prostanoids. Imaging of COX-1 and COX-2 selective radioligands with positron emission tomography (PET) may clarify how these enzymes are involved in inflammatory conditions and assist in the discovery of improved anti-inflammatory drugs. We have previously labeled the selective high-affinity COX-1 ligand, 1,5-bis(4-methoxyphenyl)-3-(2,2,2-trifluoroethoxy)-1H-1,2,4-triazole (PS13), with carbon-11 (t1/2 = 20.4 min). This radioligand ([11C]PS13) has been successful for PET imaging of COX-1 in monkey and human brain and in periphery. [11C]PS13 is being used in clinical investigations. Alternative labeling of PS13 with fluorine-18 (t1/2 = 109.8 min) is desirable to provide a longer-lived radioligand in high activity that might be readily distributed among imaging centers. However, labeling of PS13 in its 1,1,1-trifluoroethoxy group is a radiochemical challenge. Here we assess two labeling approaches based on nucleophilic addition of cyclotron-produced [18F]fluoride ion to gem-difluorovinyl precursors, either to label PS13 in one step or to produce [18F]2,2,2-trifluoroethyl p-toluenesulfonate for labeling a hydroxyl precursor. From the latter two-step approach, we obtained [18F]PS13 ready for intravenous injection in a decay-corrected radiochemical yield of 7.9% and with a molar activity of up to 7.9 GBq/μmol. PET imaging of monkey brain with [18F]PS13 shows that this radioligand can specifically image and quantify COX-1 without radiodefluorination but with some radioactivity uptake in skull, ascribed to red bone marrow. The development of a new procedure for labeling PS13 with fluorine-18 at a higher molar activity is, however, desirable to suppress occupancy of COX-1 by carrier at baseline.

Abstract Background Cyclooxygenase-2 (COX-2), which is rapidly upregulated by inflammation, is a key enzyme catalyzing the rate-limiting step in the synthesis of several inflammatory prostanoids. Successful positron emission tomography (PET) radioligand imaging of COX-2 in vivo could be a potentially powerful tool for assessing inflammatory response in the brain and periphery. To date, however, the development of PET radioligands for COX-2 has had limited success. Methods The novel PET tracer [11C]MC1 was used to examine COX-2 expression [1] in the brains of four rhesus macaques at baseline and after injection of the inflammogen lipopolysaccharide (LPS) into the right putamen, and [2] in the joints of two human participants with rheumatoid arthritis and two healthy individuals. In the primate study, two monkeys had one LPS injection, and two monkeys had a second injection 33 and 44 days, respectively, after the first LPS injection. As a comparator, COX-1 expression was measured using [11C]PS13. Results COX-2 binding, expressed as the ratio of specific to nondisplaceable uptake (BP ND) of [11C]MC1, increased on day 1 post-LPS injection; no such increase in COX-1 expression, measured using [11C]PS13, was observed. The day after the second LPS injection, a brain lesion (~ 0.5 cm in diameter) with high COX-2 density and high BP ND (1.8) was observed. Postmortem brain analysis at the gene transcript or protein level confirmed in vivo PET results. An incidental finding in an unrelated monkey found a line of COX-2 positivity along an incision in skull muscle, demonstrating that [11C]MC1 can localize inflammation peripheral to the brain. In patients with rheumatoid arthritis, [11C]MC1 successfully imaged upregulated COX-2 in the arthritic hand and shoulder and apparently in the brain. Uptake was blocked by celecoxib, a COX-2 preferential inhibitor. Conclusions Taken together, these results indicate that [11C]MC1 can image and quantify COX-2 upregulation in both monkey brain after LPS-induced neuroinflammation and in human peripheral tissue with inflammation. Trial registration ClinicalTrials.gov NCT03912428. Registered April 11, 2019.

268 Objectives: Phosphodiesterase-4 (PDE4) has emerged as a therapeutic target for central nervous system, inflammatory, and respiratory diseases. PDE4 has 4 isoforms (A-D) among which B and D are predominant in brain. Allosteric modulators for PDE4D are sought as candidate cognition enhancers and antidepressants with low potential for emesis side-effect [1]. [11C](R)-Rolipram readily images PDE4 in primate brain [2, 3]. However, [11C](R)-Rolipram is a non-subtype selective inhibitor and has no utility for showing target engagement by experimental drugs at subtypes of PDE4. A radioligand selective for imaging PDE4D with positron emission tomography (PET) would be useful for drug development and for studying PDE4D regulation in neuropsychiatric disorders. Here we describe the synthesis and evaluation of candidate radioligands ([11C]1−6 and [18F]7 for PET imaging of PDE4D in primate brain. Methods: Ligands 1−7, des-methyl precursors for labeling with carbon-11 (8−13), and an iodonium salt for fluorine-18 labeling (14) were synthesized de novo. 11C-Labeled tracers were prepared by treating a desmethyl precursor with [11C]iodomethane in the presence of a base at RT (e.g., [11C]6 and 7 in Figure 1A] or at 80 °C for 5 min. For 18F-labeling, iodonium salt precursor plus TEMPO in DMSO was added to dry[18F]fluoride ion (~ 8 GBq) and heated with microwaves (130 ⁰C for 2.5 min) [Figure 1B]. Rhesus monkeys were administered with radioligand (0.15−0.3 GBq i.v.) and PET scans were acquired on a Focus 220 scanner. The monkey was administered either rolipram (0.1, 0.2, 1 mg/kg, i.v.) or BPN14770, a selective PDE4D negative allosteric modulator (3 mg/kg, i.v.). Concurrent arterial blood sampling was performed to obtain the metabolite-corrected input function for quantification. Total distribution volumes (VT) for different regions were calculated using a two-tissue compartmental model. Results: In vitro enzyme inhibition assays revealed that 1igands 1-7 are potent (PDE4D IC50 values range subnanomolar to low nanomolar) and selective to PDE4D over PDE4B. [11C]1-6 were obtained in 10-20% decay-corrected formulated yields in ~ 40 min synthesis time with a molar activity (Am) of > 200 GBq/µmol. The yield of [18F]7 was 10% with Am of ~ 98 GBq/µmol at EOS (~ 90 min synthesis time). At baseline, each radioligand readily entered monkey brain with quick washout. However, receptor pre-block revealed no appreciable specific signal for [11C]1-[11C]4 and[18F]7. Radioligands [11C]5 and [11C]6 performed best giving moderate brain uptake (SUV ~3) and appreciable specific signal [Figure 2]. [11C]6 uptake was also appreciably blocked by BPN14770 [Figure 2C]. VT time stability of [11C]5 and [11C]6 indicated possible accumulation of radiometabolites in the brain but was adequate for quantification as the normalized VT improves towards the end of the scan (120 min). Conclusions: [11C]5 and [11C]6 are the best among the radioligands evaluated with [11C]6 being the slightly superior PET radioligand and may be used for occupancy and target engagement by experimental drugs at PDE4D in monkey.Acknowledgements: The Intramural Research Program of NIH (NIMH).References [1] Burgin AB et al., 2010Nat. Biotechnol. 28, 63. [2] Fujita M et al., 2012Biol. Psychiatry, 72, 548. [3] Ooms M et al., 2019J Cerebr Blood Flow Metab.,39, 1306.

We aimed to develop effective radioligands for quantifying brain O -linked-β- N -acetyl-glucosamine ( O -GlcNAc) hydrolase (OGA) using positron emission tomography in living subjects as tools for evaluating drug target engagement. Posttranslational modifications of tau, a biomarker of Alzheimer’s disease, by O -GlcNAc through the enzyme pair OGA and O -GlcNAc transferase (OGT) are inversely related to the amounts of its insoluble hyperphosphorylated form. Increase in tau O -GlcNAcylation by OGA inhibition is believed to reduce tau aggregation. LSN3316612, a highly selective and potent OGA ligand [half-maximal inhibitory concentration (IC 50 ) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [ 3 H]LSN3316612 imaged and quantified OGA in postmortem brains of rat, monkey, and human. The presence of fluorine and carbonyl functionality in LSN3316612 enabled labeling with positron-emitting fluorine-18 or carbon-11. Both [ 18 F]LSN3316612 and [ 11 C]LSN3316612 bound reversibly to OGA in vivo, and such binding was blocked by pharmacological doses of thiamet G, an OGA inhibitor of different chemotype, in monkeys. [ 18 F]LSN3316612 entered healthy human brain avidly (~4 SUV) without radiodefluorination or adverse effect from other radiometabolites, as evidenced by stable brain total volume of distribution (V T ) values by 110 min of scanning. Overall, [ 18 F]LSN3316612 is preferred over [ 11 C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey.

We aimed to develop radioligands for PET imaging of brain phosphodiesterase subtype 4D (PDE4D), a potential target for developing cognition enhancing or antidepressive drugs. Exploration of several chemical series gave four leads with high PDE4D inhibitory potency and selectivity, optimal lipophilicity, and good brain uptake. These leads featured alkoxypyridinyl cores. They were successfully labeled with carbon-11 (t1/2 = 20.4 min) for evaluation with PET in monkey. Whereas two of these radioligands did not provide PDE4D-specific signal in monkey brain, two others, [11C]T1660 and [11C]T1650, provided sizable specific signal, as judged by pharmacological challenge using rolipram or a selective PDE4D inhibitor (BPN14770) and subsequent biomathematical analysis. Specific binding was highest in prefrontal cortex, temporal cortex, and hippocampus, regions that are important for cognitive function. [11C]T1650 was progressed to evaluation in humans with PET, but the output measure of brain enzyme density (VT) increased with scan duration. This instability over time suggests that radiometabolite(s) were accumulating in the brain. BPN14770 blocked PDE4D uptake in human brain after a single dose, but the percentage occupancy was difficult to estimate because of the unreliability of measuring VT. Overall, these results show that imaging of PDE4D in primate brain is feasible but that further radioligand refinement is needed, most likely to avoid problematic radiometabolites.

Introduction: The serotonin 1B receptor subtype is of interest in the pathophysiology and treatment of depression, anxiety, and migraine. Over recent years 5-HT1B receptor binding in human brain has been examined with PET using radioligands that are partial but not full agonists. To explore how the intrinsic activity of a PET radioligand may affect imaging performance, two high-affinity full 5-HT-HT1B receptor agonists (AZ11136118, 4; and AZ11895987, 5) were selected from a large compound library and radiolabeled for PET examination in non-human primates. Methods: [C-11]4 was obtained through Pd(0)-mediated insertion of [C-11]carbon monoxide between prepared iodoarene and homochiral amine precursors. [C-11]5 was obtained through N-C-11-methylation of N-desmethyl precursor 6 with [C-11]methyl triflate. [C-11]4 and [C-11]5 were studied with PET in rhesus or cynomolgus monkey. [C-11]4 was studied with PET in mice and rats to measure brain uptake and specific binding. Ex-vivo experiments in rats were performed to identify whether there were radiometabolites in brain. Physiochemical parameters for (11)1C]4 (pKa, logD and conformational energetics) were evaluated. Results: Both [C-11]4 and [C-11]5 were successfully produced in high radiochemical purity and in adequate amounts for PET experiments. After intravenous injection of [C-11]4, brain radioactivity peaked at a low level (0.2 SUV). Pretreatment with tariquidar, an inhibitor of the brain P-gp efflux transporter, increased brain exposure four-fold whereas pretreatment with a high pharmacological dose of the 5-HT1B antagonist, AR-A000002, had no effect on the binding. Ex-vivo experiments in rats showed no radiometabolites entering brain. [C-11]5 also failed to enter monkey brain under baseline conditions. Conclusions: [C-11]4 and [C-11]5 show too low brain uptake and specific binding to be useful PET radioligands. Low brain uptake is partly ascribed to efflux transporter action as well as unfavorable conformations. (C) 2019 Elsevier Inc. All rights reserved.

Accumulation of hyperphosphorylated tau, a microtubule-associated protein, plays an important role in the progression of Alzheimer disease. Animal studies suggest that one strategy for treating Alzheimer disease and related tauopathies may be inhibition of O-GlcNAcase (OGA), which may subsequently decrease pathologic tau phosphorylation. Here, we report the pharmacokinetics of a novel PET radioligand, 18F-LSN3316612, which binds with high affinity and selectivity to OGA. Methods: PET imaging was performed on rhesus monkeys at baseline and after administration of either thiamet-G, a potent OGA inhibitor, or nonradioactive LSN3316612. The density of the enzyme was calculated as distribution volume using a 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. The radiation burden for future studies was based on whole-body imaging of monkeys. Oga∆Br, a mouse brain-specific knockout of Oga, was also scanned to assess the specificity of the radioligand for its target enzyme. Results: Uptake of radioactivity in monkey brain was high (∼5 SUV) and followed by slow washout. The highest uptake was in the amygdala, followed by striatum and hippocampus. Pretreatment with thiamet-G or nonradioactive LSN3316612 reduced brain uptake to a low and uniform concentration in all regions, corresponding to an approximately 90% decrease in distribution volume. Whole-body imaging of rhesus monkeys showed high uptake in kidney, spleen, liver, and testes. In Oga∆Br mice, brain uptake of 18F-LSN3316612 was reduced by 82% compared with control mice. Peripheral organs were unaffected in Oga∆Br mice, consistent with loss of OGA expression exclusively in the brain. The effective dose of 18F-LSN3316612 in humans was calculated to be 22 μSv/MBq, which is typical for 18F-labeled radioligands. Conclusion: These results show that 18F-LSN3316612 is an excellent radioligand for imaging and quantifying OGA in rhesus monkeys and mice. On the basis of these data, 18F-LSN3316612 merits evaluation in humans.

Selective high-affinity antagonists for the dopamine D3 receptor (D3R) are sought for treating substance use disorders. Positron emission tomography (PET) with an effective D3R radioligand could be a useful tool for the development of such therapeutics by elucidating pharmacological specificity and target engagement in vivo. Currently, a D3R-selective radioligand does not exist. The D3R ligand, N-(4-(4-(3-chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-1H-indole-2-carboxamide (BAK4-51, 1), has attractive properties for PET radioligand development, including full antagonist activity, very high D3R affinity, D3R selectivity, and moderate lipophilicity. We labeled 1 with the positron-emitter carbon-11 (t1/2 = 20.4 min) in the methoxy group for evaluation as a radioligand in animals with PET. However, [11C]1 was found to be an avid substrate for brain efflux transporters and lacked D3R-specific signal in rodent and monkey brain in vivo.

Cyclooxygenase (COX)-1 is generally considered a constitutive enzyme that serves a role in normal function, but studies have demonstrated that it also contributes to proinflammatory responses, particularly in brain. Developing in vivo COX-1 activity measurements would permit definitive studies in healthy and inflammatory conditions. Towards this end, our laboratory recently developed 11C-PS13, a potent and selective PET radioligand for COX-1. This study sought to determine whether 11C-PS13 can selectively image constitutive levels of COX-1 in human and nonhuman primates.

In our preceding paper (Part 1), we identified three 1,5-bis-diaryl-1,2,4-triazole-based compounds that merited evaluation as potential positron emission tomography (PET) radioligands for selectively imaging cyclooxygenase-1 (COX-1) in monkey and human brain, namely, 1,5-bis(4-methoxyphenyl)-3-(alkoxy)-1H-1,2,4-triazoles bearing a 3-methoxy (PS1), a 3-(2,2,2-trifluoroethoxy) (PS13), or a 3-fluoromethoxy substituent (PS2). PS1 and PS13 were labeled from phenol precursors by O-11C-methylation with [11C]iodomethane and PS2 by O-18F-fluoroalkylation with [2H2,18F]fluorobromomethane. Here, we evaluated these PET radioligands in monkey. All three radioligands gave moderately high uptake in brain, although [2H2,18F]PS2 also showed undesirable radioactivity uptake in skull. [11C]PS13 was selected for further evaluation, mainly based on more favorable brain kinetics than [11C]PS1. Pharmacological preblock experiments showed that about 55% of the radioactivity uptake in brain was specifically bound to COX-1. An index of enzyme density, VT, was well identified from serial brain scans and from the concentrations of parent radioligand in arterial plasma. In addition, VT values were stable within 80 min, suggesting that brain uptake was not contaminated by radiometabolites. [11C]PS13 successfully images and quantifies COX-1 in monkey brain, and merits further investigation for imaging COX-1 in monkey models of neuroinflammation and in healthy human subjects.

This study assessed whether the newly developed PET radioligands 11C-PS13 and 11C-MC1 could image constitutive levels of cyclooxygenase (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 2 radioligands were also performed after administration of a nonradioactive drug preferential for either COX-1 or COX-2. Concurrent venous samples were obtained to measure parent radioligand concentrations. SUVs were calculated from 10 to 90 min. 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.