In Vivo Evaluation of 6 Analogs of 11C-ER176 as Candidate 18F-Labeled Radioligands for 18-kDa Translocator Protein.

Because of its excellent ratio of specific to nondisplaceable uptake, the radioligand 11C-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 18F-labeled TSPO PET radioligand based on ER176 with the potential for broader distribution. This study used generic 11C 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 18F. Methods: Six fluorine-containing analogs of ER176-3 fluoro and 3 trifluoromethyl isomers-were synthesized and labeled by 11C 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 (VT), an indirect measure of lack of radiometabolite accumulation in the brain. Results: Total TSPO binding was quantified as VT corrected for plasma free fraction (VT/fP) using Logan graphical analysis for all 6 radioligands. VT/fP 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, VT 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 VT Among them, the o-fluoro, m-trifluoromethyl, and m-fluoro compounds were the 3 best candidates for development as radioligands with an 18F label.

Synthesis and Screening in Mice of Fluorine-Containing PET Radioligands for TSPO: Discovery of a Promising 18F-Labeled Ligand.

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.

[11C]deschloroclozapine is an improved PET radioligand for quantifying a human muscarinic DREADD expressed in monkey brain.

Previous work found that [11C]deschloroclozapine ([11C]DCZ) is superior to [11C]clozapine ([11C]CLZ) for imaging Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). This study used PET to quantitatively and separately measure the signal from transfected receptors, endogenous receptors/targets, and non-displaceable binding in other brain regions to better understand this superiority. A genetically-modified muscarinic type-4 human receptor (hM4Di) was injected into the right amygdala of a male rhesus macaque. [11C]DCZ and [11C]CLZ PET scans were conducted 2-24 months later. Uptake was quantified relative to the concentration of parent radioligand in arterial plasma at baseline (n = 3 scans/radioligand) and after receptor blockade (n = 3 scans/radioligand). Both radioligands had greater uptake in the transfected region and displaceable uptake in other brain regions. Displaceable uptake was not uniformly distributed, perhaps representing off-target binding to endogenous receptor(s). After correction, [11C]DCZ signal was 19% of that for [11C]CLZ, and background uptake was 10% of that for [11C]CLZ. Despite stronger [11C]CLZ binding, the signal-to-background ratio for [11C]DCZ was almost two-fold greater than for [11C]CLZ. Both radioligands had comparable DREADD selectivity. All reference tissue models underestimated signal-to-background ratio in the transfected region by 40%-50% for both radioligands. Thus, the greater signal-to-background ratio of [11C]DCZ was due to its lower background uptake.

Synthesis of [18F]PS13 and Evaluation as a PET Radioligand for Cyclooxygenase-1 in Monkey.

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.

PET measurement of cyclooxygenase-2 using a novel radioligand: upregulation in primate neuroinflammation and first-in-human study.

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 (BPND) 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 BPND (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.

PET ligands [18F]LSN3316612 and [11C]LSN3316612 quantify O-linked-ß-N-acetyl-glucosamine hydrolase in the brain.

We aimed to develop effective radioligands for quantifying brain O-linked-ß-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 (IC50) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [3H]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 [18F]LSN3316612 and [11C]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. [18F]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 (VT) values by 110 min of scanning. Overall, [18F]LSN3316612 is preferred over [11C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey.

Discovery, Radiolabeling, and Evaluation of Subtype-Selective Inhibitors for Positron Emission Tomography Imaging of Brain Phosphodiesterase-4D.

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.

Synthesis and evaluation of two new candidate high-affinity full agonist PET radioligands for imaging 5-HT1B receptors.

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-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: [11C]4 was obtained through Pd(0)-mediated insertion of [11C]carbon monoxide between prepared iodoarene and homochiral amine precursors. [11C]5 was obtained through N-11C-methylation of N-desmethyl precursor 6 with [11C]methyl triflate. [11C]4 and [11C]5 were studied with PET in rhesus or cynomolgus monkey. [11C]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 [11C]4 (pKa, logD and conformational energetics) were evaluated. RESULTS: Both [11C]4 and [11C]5 were successfully produced in high radiochemical purity and in adequate amounts for PET experiments. After intravenous injection of [11C]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. [11C]5 also failed to enter monkey brain under baseline conditions. CONCLUSIONS: [11C]4 and [11C]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.

Evaluation of a PET Radioligand to Image O-GlcNAcase in Brain and Periphery of Rhesus Monkey and Knock-Out Mouse.

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.

[O-methyl-11C]N-(4-(4-(3-Chloro-2-methoxyphenyl)-piperazin-1-yl)butyl)-1H-indole-2-carboxamide ([11C]BAK4-51) Is an Efflux Transporter Substrate and Ineffective for PET Imaging of Brain D3 Receptors in Rodents and Monkey.

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.

Evaluation of Two Potent and Selective PET Radioligands to Image COX-1 and COX-2 in Rhesus Monkeys.

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.

3-Substituted 1,5-Diaryl-1 H-1,2,4-triazoles as Prospective PET Radioligands for Imaging Brain COX-1 in Monkey. Part 2: Selection and Evaluation of [11C]PS13 for Quantitative Imaging.

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)-1 H-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.

The PET Radioligand 18F-FIMX Images and Quantifies Metabotropic Glutamate Receptor 1 in Proportion to the Regional Density of Its Gene Transcript in Human Brain.

UNLABELLED: A recent study from our laboratory found that (18)F-FIMX is an excellent PET radioligand for quantifying metabotropic glutamate receptor 1 (mGluR1) in monkey brain. This study evaluated the ability of (18)F-FIMX to quantify mGluR1 in humans. A second goal was to use the relative density of mGluR1 gene transcripts in brain regions to estimate specific uptake and nondisplaceable uptake (VND) in each brain region. METHODS: After injection of 189 ± 3 MBq of (18)F-FIMX, 12 healthy volunteers underwent a dynamic PET scan over 120 min. For 6 volunteers, images were acquired until 210 min. A metabolite-corrected arterial input function was measured from the radial artery. Four other subjects underwent whole-body scanning to estimate radiation exposure. RESULTS: (18)F-FIMX uptake into the human brain was high (SUV = 4-6 in the cerebellum), peaked at about 10 min, and washed out rapidly. An unconstrained 2-tissue-compartment model fitted the data well, and distribution volume (VT) (mL⋅cm(-3)) values ranged from 1.5 in the caudate to 11 in the cerebellum. A 120-min scan provided stable VT values in all regions except the cerebellum, for which an acquisition time of at least 170 min was necessary. VT values in brain regions correlated well with mGluR1 transcript density, and the correlation suggested that VND of (18)F-FIMX was quite low (0.5 mL⋅cm(-3)). This measure of VND in humans was similar to that from a receptor blocking study in monkeys, after correcting for differences in plasma protein binding. Similar to other (18)F-labeled ligands, the effective dose was about 23 µSv/MBq. CONCLUSION: (18)F-FIMX can quantify mGluR1 in the human brain with a 120- to 170-min scan. Correlation of brain uptake with the relative density of mGluR1 transcript allows specific receptor binding of a radioligand to be quantified without injecting pharmacologic doses of a blocking agent.

[carbonyl-11C]4-Fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide ([11C]FIMX) is an effective radioligand for PET imaging of metabotropic glutamate receptor 1 (mGluR1) in monkey brain.

INTRODUCTION: Metabotropic glutamate subtype receptor 1 (mGluR1) is implicated in several neuropsychiatric disorders and is a target for drug development. [(18)F]FIMX ([(18)F]4-fluoro--N-methyl-N--(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide) is an effective radioligand for imaging brain mGluR1 with PET. A similarly effective radioligand with a shorter half-life would usefully allow PET studies of mGluR1 at baseline and after pharmacological or other challenge on the same day. Here we describe the preparation of [(11)C]FIMX for evaluation in monkey with PET. METHODS: [(11)C]FIMX was prepared via Pd-promoted carbonylation of 1-fluoro-4-iodobenzene with [(11)C]carbon monoxide, aminolysis of the [(11)C]acyl-palladium complex with the requisite Boc-protected amine, and deprotection with HCl in THF. PET scans of [(11)C]FIMX injected into a monkey were performed at baseline and after preblock of mGluR1 with measurement of the arterial input function. RESULTS: The radiosynthesis required 42 min and gave [(11)C]FIMX in about 5% overall decay-corrected radiochemical yield and with a specific activity of about 100 GBq/µmol. PET in rhesus monkey at baseline showed that radioactivity peaked high in receptor-rich cerebellum and much lower in receptor-poor occipital cortex. Radioactivity in cerebellum declined to 32% of peak at 85 min. VT at baseline appeared stable in all brain regions after 60 min. Under mGluR1 pre-blocked condition, radioactivity uptake in all regions declined more rapidly to a low level. Receptor pre-block reduced VT from 13.0 to 1.5 in cerebellum and from 2.9 to 1.4 in occipital cortex. CONCLUSION: [(11)C]FIMX is an effective radioligand for imaging mGluR1 in monkey with PET.

Synthesis and evaluation of translocator 18 kDa protein (TSPO) positron emission tomography (PET) radioligands with low binding sensitivity to human single nucleotide polymorphism rs6971.

The imaging of translocator 18 kDa protein (TSPO) in living human brain with radioligands by positron emission tomography (PET) has become an important means for the study of neuroinflammatory conditions occurring in several neuropsychiatric disorders. The widely used prototypical PET radioligand [(11)C](R)-PK 11195 ([(11)C](R)-1; [N-methyl-(11)C](R)-N-sec-butyl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide) gives a low PET signal and is difficult to quantify, whereas later generation radioligands have binding sensitivity to a human single nucleotide polymorphism (SNP) rs6971, which imposes limitations on their utility for comparative quantitative PET studies of normal and diseased subjects. Recently, azaisosteres of 1 have been developed with improved drug-like properties, including enhanced TSPO affinity accompanied by moderated lipophilicity. Here we selected three of these new ligands (7-9) for labeling with carbon-11 and for evaluation in monkey as candidate PET radioligands for imaging brain TSPO. Each radioligand was readily prepared by (11)C-methylation of an N-desmethyl precursor and was found to give a high proportion of TSPO-specific binding in monkey brain. One of these radioligands, [(11)C]7, the direct 4-azaisostere of 1, presents many radioligand properties that are superior to those reported for [(11)C]1, including higher affinity, lower lipophilicity, and stable quantifiable PET signal. Importantly, 7 was also found to show very low sensitivity to the human SNP rs6971 in vitro. Therefore, [(11)C]7 now warrants evaluation in human subjects with PET to assess its utility for imaging TSPO in human brain, irrespective of subject genotype.

Evaluation in monkey of two candidate PET radioligands, [11 C]RX-1 and [18 F]RX-2, for imaging brain 5-HT4 receptors.

The serotonin subtype-4 (5-HT4 ) receptor, which is known to be involved physiologically in learning and memory, and pathologically in Alzheimer's disease, anxiety, and other neuropsychiatric disorders-has few radioligands readily available for imaging in vivo. We have previously reported two novel 5-HT4 receptor radioligands, namely [methoxy-11 C](1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [11 C]RX-1), and the [18 F]3-fluoromethoxy analog ([18 F]RX-2), and in this study we evaluated them by PET in rhesus monkey. Brain scans were performed at baseline, receptor preblock or displacement conditions using SB 207710, a 5-HT4 receptor antagonist, on the same day for [11 C]RX-1 and on different days for [18 F]RX-2. Specific-to-nondisplaceable ratio (BPND ) was measured with the simplified reference tissue model from all baseline scans. To determine specific binding, total distribution volume (VT ) was also measured in some monkeys by radiometabolite-corrected arterial input function after ex vivo inhibition of esterases from baseline and blocked scans. Both radioligands showed moderate to high peak brain uptake of radioactivity (2-6 SUV). Regional BPND values were in the rank order of known 5-HT4 receptor distribution with a trend for higher BPND values from [18 F]RX-2. One-tissue compartmental model provided good fits with well identified VT values for both radioligands. In the highest 5-HT4 receptor density region, striatum, 50-60% of total binding was specific. The VT in receptor-poor cerebellum reached stable values by about 60 min for both radioligands indicating little influence of radiometabolites on brain signal. In conclusion, both [11 C]RX-1 and [18 F]RX-2 showed positive attributes for PET imaging of brain 5-HT4 receptors, validating the radioligand design strategy. Synapse 68:613-623, 2014. © 2014 Wiley Periodicals, Inc.

(11)C-CUMI-101, a PET radioligand, behaves as a serotonin 1A receptor antagonist and also binds to α(1) adrenoceptors in brain.

UNLABELLED: The PET radioligand (11)C-CUMI-101 was previously suggested as a putative agonist radioligand for the serotonin 1A (5-hydroxytryptamine 1A [5-HT1A]) receptor in recombinant cells expressing human 5-HT1A receptor. However, a recent study showed that CUMI-101 behaved as a potent 5-HT1A receptor antagonist in rat brain. CUMI-101 also has moderate affinity (Ki = 6.75 nM) for α1 adrenoceptors measured in vitro. The current study examined the functional properties and selectivity of CUMI-101, both in vitro and in vivo. METHODS: The functional assay was performed using (35)S-GTPγS (GTP is guanosine triphosphate) in primate brains. The cross-reactivity of CUMI-101 with α1 adrenoceptors was performed using in vitro radioligand binding studies in rat, monkey, and human brains as well as in vivo PET imaging in mouse, rat, and monkey brains. RESULTS: CUMI-101 did not stimulate (35)S-GTPγS binding in primate brain, in contrast to 8-OH-DPAT, a potent 5-HT1A receptor agonist. Instead, CUMI-101 behaved as a potent 5-HT1A receptor antagonist by dose-dependently inhibiting 8-OH-DPAT-stimulated (35)S-GTPγS binding. Both in vitro and in vivo studies showed that CUMI-101 had significant α1 adrenoceptor cross-reactivity. On average, across all 3 species examined, cross-reactivity was highest in the thalamus (>45%) and lowest in the neocortex and cerebellum (<10%). PET imaging further confirmed that only preblocking with WAY-100635 plus prazosin decreased (11)C-CUMI-101 brain uptake to that of self-block. CONCLUSION: CUMI-101 behaves as a 5-HT1A receptor antagonist in primate brain, with significant, regional-dependent α1 adrenoceptor cross-reactivity, limiting its potential use as a PET radioligand in humans.

In vitro and in vivo evaluation of (11)C-SD5024, a novel PET radioligand for human brain imaging of cannabinoid CB1 receptors.

We recently developed a novel cannabinoid subtype-1 (CB1) receptor radioligand (11)C-SD5024 for brain imaging. This study aimed to evaluate (11)C-SD5024 both in vitro and in vivo and compare it with the other CB1 receptor ligands previously used in humans, i.e., (11)C-MePPEP, (11)C-OMAR, (18)F-MK-9470, and (18)F-FMPEP-d2. In vitro experiments were performed to measure dissociation constant (Ki) in the human brain and to measure the lipophilicity of the five CB1 receptor ligands listed above. In vivo specific binding in monkeys was measured by comparing total distribution volume (VT) at baseline and after full receptor blockade. The kinetics of (11)C-SD5024 in humans were evaluated in seven healthy subjects with compartmental modeling. SD5024 showed Ki=0.47nM, which was at an intermediate level among the five CB1 receptor ligands. Lipophilicity (LogD7.4) was 3.79, which is appropriate for brain imaging. Monkey scans showed high proportion of specific binding: ~80% of VT. In humans, (11)C-SD5024 showed peak brain uptake of 1.5-3 standardized uptake value, which was slightly higher than that of (11)C-OMAR and (18)F-MK-9470. One-compartment model showed good fitting, consistent with the vast majority of brain uptake being specific binding found in the monkey. Regional VT values were consistent with known distribution of CB1 receptors. VT calculated from 80 and 120min of scan data was strongly correlated (R(2)=0.97), indicating that 80min provided adequate information for quantitation and that the influence of radiometabolites was low. Intersubject variability for VT of (11)C-SD5024 was 22%, which was low among the five radioligands and indicated precise measurement. In conclusion, (11)C-SD5024 has appropriate affinity and lipophilicity, high specific binding, moderate brain uptake, and provides good precision to measure the binding. The results suggest that (11)C-SD5024 is slightly better than or equivalent to (11)C-OMAR and that both are suitable for clinical studies, especially those that involve two scans in one day.

Synthesis and evaluation in monkey of [(18)F]4-fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide ([(18)F]FIMX): a promising radioligand for PET imaging of brain metabotropic glutamate receptor 1 (mGluR1).

We sought to develop a PET radioligand that would be useful for imaging human brain metabotropic subtype 1 receptors (mGluR1) in neuropsychiatric disorders and in drug development. 4-Fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide (FIMX, 11) was identified as having favorable properties for development as a PET radioligand. We developed a method for preparing [(18)F]11 in useful radiochemical yield and in high specific activity from [(18)F]fluoride ion and an N-Boc-protected (phenyl)aryliodonium salt precursor (15). In baseline experiments in rhesus monkey, [(18)F]11 gave high brain radioactivity uptake, reflecting the expected distribution of mGluR1 with notably high uptake in cerebellum, which became 47% lower by 120 min after radioligand injection. Pharmacological challenges demonstrated that a very high proportion of the radioactivity in monkey brain was bound specifically and reversibly to mGluR1. [(18)F]11 is concluded to be an effective PET radioligand for imaging mGluR1 in monkey brain and therefore merits further evaluation in human subjects.

11C-LY2428703, a positron emission tomographic radioligand for the metabotropic glutamate receptor 1, is unsuitable for imaging in monkey and human brains.

BACKGROUND: A recent study from our laboratory demonstrated that 11C-LY2428703, a new positron emission tomographic radioligand for metabotropic glutamate receptor 1 (mGluR1), has promising in vitro properties and excellent in vivo performance for imaging rat brain. The present study evaluated 11C-LY2428703 for imaging mGluR1 in monkey and human brains. METHODS: Rhesus monkeys were imaged at baseline and after administration of an mGluR1 blocking agent to calculate nonspecific binding, as well as after the administration of permeability glycoprotein (P-gp) and breast cancer resistance protein (BCRP) blockers to assess whether 11C-LY2428703 is a substrate for efflux transporters at the blood-brain barrier. Human imaging was performed at baseline in three healthy volunteers, and arterial input function was measured. RESULTS: Overall brain uptake was low in monkeys, though slightly higher in the cerebellum, where mGluR1s are concentrated. However, the uptake was not clearly displaceable in the scans after mGluR1 blockade. Brain penetration of the ligand did not increase after P-gp and BCRP blockade. Brain uptake was similarly low in all human subjects (mean VT with a two-tissue compartment model, 0.093 ± 0.012 mL/cm3) and for all regions, including the cerebellum. CONCLUSIONS: Despite promising in vitro and in vivo results in rodents, 11C-LY2428703 was unsuitable for imaging mGluR1s in monkey or human brain because of low brain uptake, which was likely caused by high binding to plasma proteins.