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.

[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.

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.

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.

Synthesis and characterization in monkey of [11C]SP203 as a radioligand for imaging brain metabotropic glutamate 5 receptors.

PURPOSE: [(18)F]SP203 (3-fluoro-5-(2-(2-([(18)F]fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile) is an effective high-affinity and selective radioligand for imaging metabotropic 5 receptors (mGluR5) in human brain with PET. To provide a radioligand that may be used for more than one scanning session in the same subject in a single day, we set out to label SP203 with shorter-lived (11)C (t (1/2) = 20.4 min) and to characterize its behavior as a radioligand with PET in the monkey. METHODS: Iodo and bromo precursors were obtained by cross-coupling 2-fluoromethyl-4-((trimethylsilyl)ethynyl)-1,3-thiazole with 3,5-diiodofluorobenzene and 3,5-dibromofluorobenzene, respectively. Treatment of either precursor with [(11)C]cyanide ion rapidly gave [(11)C]SP203, which was purified with high-performance liquid chromatography. PET was used to measure the uptake of radioactivity in brain regions after injecting [(11)C]SP203 intravenously into rhesus monkeys at baseline and under conditions in which mGluR5 were blocked with 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP). The emergence of radiometabolites in monkey blood in vitro and in vivo was assessed with radio-HPLC. The stability of [(11)C]SP203 in human blood in vitro was also measured. RESULTS: The iodo precursor gave [(11)C]SP203 in higher radiochemical yield (>98 %) than the bromo precursor (20-52 %). After intravenous administration of [(11)C]SP203 into three rhesus monkeys, radioactivity peaked early in brain (average 12.5 min) with a regional distribution in rank order of expected mGluR5 density. Peak uptake was followed by a steady decline. No radioactivity accumulated in the skull. In monkeys pretreated with MTEP before [(11)C]SP203 administration, radioactivity uptake in brain was again high but then declined more rapidly than in the baseline scan to a common low level. [(11)C]SP203 was unstable in monkey blood in vitro and in vivo, and gave predominantly less lipophilic radiometabolites. By contrast, [(11)C]SP203 was stable in human blood in vitro. CONCLUSION: [(11)C]SP203 emulates [(18)F]SP203 with regard to providing a sizeable mGluR5-specific signal in monkey brain, and advantageously avoids troublesome accumulation of radioactivity in bone. Although [(11)C]SP203 is unsuitable for mGluR5 quantification in monkey brain, its evaluation as a PET radioligand for studying human brain mGluR5 is nevertheless warranted.

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.

Kinetic analysis in human brain of [11C](R)-rolipram, a positron emission tomographic radioligand to image phosphodiesterase 4: a retest study and use of an image-derived input function.

UNLABELLED: [(11)C](R)-rolipram provides a measure of the density of phosphodiesterase 4 (PDE4) in brain, an enzyme that metabolizes cAMP. The aims of this study were to perform kinetic modeling of [(11)C](R)-rolipram in healthy humans using an arterial input function and to replace this arterial input in humans with an image-derived input function. METHODS: Twelve humans had two injections of [(11)C](R)-rolipram. An image-derived input function was obtained from the carotid arteries and four blood samples. The samples were used for partial volume correction and for estimating the parent concentration using HPLC analysis. RESULTS: An unconstrained two-compartment model and Logan analysis measured distribution volume V(T), with good identifiability but with moderately high retest variability (15%). Similar results were obtained using the image input (ratio image/arterial V(T)=1.00±0.06). CONCLUSIONS: Binding of [(11)C](R)-rolipram to PDE4 can be quantified in human brain using kinetic modeling and an arterial input function. Image input function from carotid arteries provides an equally accurate and reproducible method to quantify PDE4.

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.

[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 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.

Comparison of [(11)C]-(R)-PK 11195 and [(11)C]PBR28, two radioligands for translocator protein (18 kDa) in human and monkey: Implications for positron emission tomographic imaging of this inflammation biomarker.

UNLABELLED: Ten percent of humans lack specific binding of [(11)C]PBR28 to 18 kDa translocator protein (TSPO), a biomarker for inflammation. "Non-binders" have not been reported using another TSPO radioligand, [(11)C]-(R)-PK 11195, despite its use for more than two decades. This study asked two questions: (1) What is the cause of non-binding to PBR28? and (2) Why has this phenomenon not been reported using [(11)C]-(R)-PK 11195? METHODS: Five binders and five non-binders received whole-body imaging with both [(11)C]-(R)-PK 11195 and [(11)C]PBR28. In vitro binding was performed using leukocyte membranes from binders and non-binders and the tritiated versions of the ligand. Rhesus monkeys were imaged with [(11)C]-(R)-PK 11195 at baseline and after blockade of TSPOs. RESULTS: Using [(11)C]PBR28, uptake in all five organs with high densities of TSPO (lung, heart, brain, kidney, and spleen) was 50% to 75% lower in non-binders than in binders. In contrast, [(11)C]-(R)-PK 11195 distinguished binders and non-binders in only heart and lung. For the in vitro assay, [(3)H]PBR28 had more than 10-fold lower affinity to TSPO in non-binders than in binders. The in vivo specific binding of [(11)C]-(R)-PK 11195 in monkey brain was approximately 80-fold lower than that reported for [(11)C]PBR28. CONCLUSIONS: Based on binding of [(3)H]PK 11195 to leukocyte membranes, both binders and non-binders express TSPO. Non-binding to PBR28 is caused by its low affinity for TSPO in non-binders. Non-binding may be differentially expressed in organs of the body. The relatively low in vivo specific binding of [(11)C]-(R)-PK 11195 may have obscured its detection of non-binding in peripheral organs.

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.

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.

Human brain imaging and radiation dosimetry of 11C-N-desmethyl-loperamide, a PET radiotracer to measure the function of P-glycoprotein.

UNLABELLED: P-glycoprotein (P-gp) is a membrane-bound efflux pump that limits the distribution of drugs to several organs of the body. At the blood-brain barrier, P-gp blocks the entry of both loperamide and its metabolite, N-desmethyl-loperamide (N-dLop), and thereby prevents central opiate effects. Animal studies have shown that (11)C-dLop, compared with (11)C-loperamide, is an especially promising radiotracer because it generates negligible radiometabolites that enter the brain. The purposes of this study were to determine whether (11)C-dLop is a substrate for P-gp at the blood-brain barrier in humans and to measure the distribution of radioactivity in the entire body to estimate radiation exposure. METHODS: Brain PET scans were acquired in 4 healthy subjects for 90 min and included concurrent measurements of the plasma concentration of unchanged radiotracer. Time-activity data from the whole brain were quantified using a 1-tissue-compartment model to estimate the rate of entry (K(1)) of radiotracer into the brain. Whole-body PET scans were acquired in 8 healthy subjects for 120 min. RESULTS: For brain imaging, after the injection of (11)C-dLop the concentration of radioactivity in the brain was low (standardized uptake value, approximately 15%) and stable after approximately 20 min. In contrast, uptake of radioactivity in the pituitary was about 50-fold higher than that in the brain. The plasma concentration of (11)C-dLop declined rapidly, but the percentage composition of plasma was unusually stable, with the parent radiotracer constituting 85% of total radioactivity after approximately 5 min. The rate of brain entry was low (K(1) = 0.009 +/- 0.002 mL.cm(-3).min(-1); n = 4). For whole-body imaging, as a measure of radiation exposure to the entire body the effective dose of (11)C-dLop was 7.8 +/- 0.6 muSv/MBq (n = 8). CONCLUSION: The low brain uptake of radioactivity is consistent with (11)C-dLop being a substrate for P-gp in humans and confirms that this radiotracer generates negligible quantities of brain-penetrant radiometabolites. In addition, the low rate of K(1) is consistent with P-gp rapidly effluxing substrates while they transit through the lipid bilayer. The radiation exposure of (11)C-dLop is similar to that of many other (11)C-radiotracers. Thus, (11)C-dLop is a promising radiotracer to study the function of P-gp at the blood-brain barrier, at which impaired function would allow increased uptake into the brain.

P-glycoprotein function at the blood-brain barrier imaged using 11C-N-desmethyl-loperamide in monkeys.

UNLABELLED: 11C-Loperamide is an avid substrate for P-glycoprotein (P-gp), but it is rapidly metabolized to 11C-N-desmethyl-loperamide (11C-dLop), which is also a substrate for P-gp and thereby contaminates the radioactive signal in the brain. Should further demethylation of 11C-dLop occur, radiometabolites with low entry into the brain are generated. Therefore, we evaluated the ability of 11C-dLop to quantify the function of P-gp at the blood-brain barrier in monkeys. METHODS: Six monkeys underwent 12 PET scans of the brain, 5 at baseline and 7 after pharmacologic blockade of P-gp. A subset of monkeys also underwent PET scans with 15O-water to measure cerebral blood flow. To determine whether P-gp blockade affected peripheral distribution of 11C-dLop, we measured whole-body biodistribution in 4 monkeys at baseline and after P-gp blockade. RESULTS: The concentration of 11C-dLop in the brain was low under baseline conditions and increased 5-fold after P-gp blockade. This increase was primarily caused by an increased rate of entry into the brain rather than a decreased rate of removal from the brain. With P-gp blockade, uptake of radioactivity among brain regions correlated linearly with blood flow, suggesting a high single-pass extraction. After correction for cerebral blood flow, the uptake of 11C-dLop was fairly uniform among brain regions, suggesting that the function of P-gp is fairly uniformly distributed in the brain. On whole-body imaging, P-gp blockade significantly affected distribution of radioactivity only to the brain and not to other visually identified source organs. The effective dose estimated for humans was approximately 9 microSv/MBq. CONCLUSION: PET with 11C-dLop can quantify P-gp function at the blood-brain barrier in monkeys. The single-pass extraction of 11C-dLop is high and requires correction for blood flow to accurately measure the function of this efflux transporter. The low uptake at baseline and markedly increased uptake after P-gp blockade suggest that 11C-dLop will be useful to measure a wide range of P-gp functions at the blood-brain barrier in humans.

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.

The PET radioligand [11C]MePPEP binds reversibly and with high specific signal to cannabinoid CB1 receptors in nonhuman primate brain.

The cannabinoid CB(1) receptor is one of the most abundant G protein-coupled receptors in the brain and is a promising target of therapeutic drug development. Success of drug development for neuropsychiatric indications is significantly enhanced with the ability to directly measure spatial and temporal binding of compounds to receptors in central compartments. We assessed the utility of a new positron emission tomography (PET) radioligand to image CB(1) receptors in monkey brain. [(11)C]MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) has high CB(1) affinity (K(b)=0.574+/-0.207 nM) but also moderately high lipophilicity (measured LogD(7.4)=4.8). After intravenous injection of [(11)C]MePPEP, brain activity reached high levels of almost 600% standardized uptake value (SUV) within 10-20 min. The regional uptake was consistent with the distribution of CB(1) receptors, with high radioactivity in striatum and cerebellum and low in thalamus and pons. Injection of pharmacological doses of CB(1)-selective agents confirmed that the tracer doses of [(11)C]MePPEP reversibly labeled CB(1) receptors. Preblockade or displacement with two CB(1) selective agents (ISPB; (4-(3-cyclopentyl-indole-1-sulfonyl)-N-(tetrahydro-pyran-4-ylmethyl)-benzamide) and rimonabant) showed that the majority (>89%) of brain uptake in regions with high receptor densities was specific and reversibly bound to CB(1) receptors in the high binding regions. [(11)C]MePPEP was rapidly removed from arterial plasma. Regional brain uptake could be quantified as distribution volume relative to the concentration of parent radiotracer in plasma. The P-glycoprotein (P-gp) inhibitor DCPQ ((R)-4-[(1a,6,10b)-1,1-dichloro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol) did not significantly increase brain uptake of [(11)C]MePPEP, suggesting it is not a substrate for this efflux transporter at the blood-brain barrier. [(11)C]MePPEP is a radioligand with high brain uptake, high specific signal to CB(1) receptors, and adequately fast washout from brain that allows quantification with (11)C (half-life=20 min). These promising results in monkey justify studying this radioligand in human subjects.

11C-loperamide and its N-desmethyl radiometabolite are avid substrates for brain permeability-glycoprotein efflux.

UNLABELLED: Loperamide, an opiate receptor agonist, does not cross the blood-brain barrier because it is a substrate for the permeability-glycoprotein (P-gp) efflux pump. We evaluated 11C-loperamide as a PET radiotracer to measure P-gp function in vivo. METHODS: Monkeys were injected with 11C-loperamide, and PET brain images were acquired for 120 min. The baseline scans were followed by scans acquired after administration of either of 2 P-gp inhibitors, (2R)-anti-5-{3-[4-(10,11-dichloromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (DCPQ) or tariquidar. Both the PET scans and ex vivo measurements were obtained in P-gp knockout and wild-type mice. RESULTS: Pharmacologic inhibition of P-gp in monkeys dose-dependently increased brain activity, with a 3.7-fold effect at the highest DCPQ dose (8 mg/kg intravenously). This increase of brain activity was not caused peripherally, because DCPQ insignificantly changed the plasma concentration and plasma protein binding of radiotracer. Furthermore, the structurally dissimilar inhibitor, tariquidar, also increased brain uptake with potency equal to that of DCPQ. P-gp knockout mice had 3-fold higher brain activity on PET than did wild-type animals. Four radiometabolites were detected in the plasma and brains of ex vivo mice. The most lipophilic radiometabolite was found to be comobile with reference dLop on high-performance liquid chromatography. The brain concentrations of 11C-loperamide and the putative 11C-dLop were about 16-fold greater in P-gp knockout mice than in wild-type mice. CONCLUSION: Both 11C-loperamide and its putative radiometabolite 11C-dLop are avid P-gp substrates. 11C-dLop may be superior to 11C-loperamide in measuring P-gp function at the blood-brain barrier, because further demethylation of 11C-dLop will generate radiometabolites that have little entry into the brain.

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.