Radiosynthesis and ex vivo evaluation of [(18)F]-(S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one for imaging MAO-B with PET.

Carbon-11 labeled SL25.1188 ((S)-5-(methoxymethyl)-3-(6-(4,4,4-trifluorobutoxy)benzo[d]isoxazol-3-yl)oxazolidin-2-one) is a reversible radiotracer for monoamine oxidase B that was recently evaluated in healthy volunteers by positron emission tomography (PET). Herein we report the preparation and ex vivo evaluation of a fluorinated SL25.1188 derivative as a candidate (18)F-labeled PET radiotracer. (S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxy methyl)oxazolidin-2-one (1) was labeled with fluorine-18 in 51% uncorrected radiochemical yield having high radiochemical purity (>98%) and specific activity (109±26GBq/µmol). Ex vivo biodistribution studies demonstrated low radioactivity retention, specific binding and metabolic stability within rat brains. High uptake of radioactivity in bone is consistent with metabolic defluorination. In vitro binding assays of longer chain fluoroalkoxy derivatives revealed that the length of the carbon chain is an integral feature in MAO-B inhibitor potency and selectivity within this scaffold.

Radiosynthesis and ex vivo evaluation of [(11)C-carbonyl]carbamate- and urea-based monoacylglycerol lipase inhibitors.

INTRODUCTION: Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are the two primary enzymes that regulate the tone of endocannabinoid signaling. Although new PET radiotracers have been discovered for imaging FAAH in vivo, no such radiotracer exists for imaging MAGL. Here we report the radiosynthesis of five candidate MAGL radiotracers and their ex vivo evaluations in mice and rats. METHODS: Candidate carbamate and urea MAGL inhibitors were radiolabeled at the carbonyl position by [(11)C]CO2 fixation. Radiotracers were administered (tail-vein injection) to rodents and brain uptake of radioactivity measured at early and late time points ex vivo. Specificity of uptake was explored by pretreatment with unlabeled inhibitors (2 mg/kg, ip) 30 min prior to radiotracer administration. RESULTS: All five candidate MAGL radiotracers were prepared in high specific activity (>65 GBq/µmol) and radiochemical purity (>98%). Moderate brain uptake (0.2-0.8 SUV) was observed for each candidate while pretreatment did not reduce uptake for four of the five tested. For two candidates ([(11)C]12 and [(11)C]14), high retention of radioactivity was observed in the blood (ca. 10 and 4 SUV at 40 min) which was blocked by pretreatment with unlabeled inhibitors. The most promising candidate, [(11)C]18, demonstrated moderate brain uptake (ca. 0.8 SUV) which showed circa 50% blockade by pretreatment with unlabeled 18. CONCLUSION: One putative and four reported potent and selective MAGL inhibitors have been radiolabeled via [(11)C]CO2 fixation as radiotracers for this enzyme. Despite the promising in vitro pharmacological profile, none of the five candidate radiotracers exhibited in vivo behavior suitable for PET neuroimaging.

Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase.

INTRODUCTION: Fatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [(11)C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor. METHODS: PF-04457845 was radiolabeled at the carbonyl position via automated [(11)C]CO(2)-fixation. Ex vivo brain biodistribution of [(11)C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [(11)C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma. RESULTS: The title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [(11)C]CO(2) (decay uncorrected) within 25 min from end-of-bombardment in >98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/µmol at end-of-synthesis. Uptake of [(11)C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2-4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71-81%, demonstrating specificity for FAAH. The binding of [(11)C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted. CONCLUSIONS: [(11)C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in conscious rodents demonstrate that [11C PF-04457845 is a promising candidate radiotracer for imaging FAAH in the brain with PET. These results coupled with the known pharmacology and toxicology of PF-04457845 should facilitate clinical translation of this radiotracer.

Development and characterization of a promising fluorine-18 labelled radiopharmaceutical for in vivo imaging of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH), the enzyme responsible for terminating signaling by the endocannabinoid anandamide, plays an important role in the endocannabinoid system, and FAAH inhibitors are attractive drugs for pain, addiction, and neurological disorders. The synthesis, radiosynthesis, and evaluation, in vitro and ex vivo in rat, of an (18)F-radiotracer designed to image FAAH using positron emission tomography (PET) is described. Fluorine-18 labelled 3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate, [(18)F]5, was synthesized at high specific activity in a one-pot three step reaction using a commercial module with a radiochemical yield of 17-22% (from [(18)F]fluoride). In vitro assay using rat brain homogenates showed that 5 inhibited FAAH in a time-dependent manner, with an IC50 value of 0.82nM after a preincubation of 60min. Ex vivo biodistribution studies and ex vivo autoradiography in rat brain demonstrated that [(18)F]5 had high brain penetration with standard uptake values of up to 4.6 and had a regional distribution which correlated with reported regional FAAH enzyme activity. Specificity of binding to FAAH with [(18)F]5 was high (>90%) as demonstrated by pharmacological challenges with potent and selective FAAH inhibitors and was irreversible as demonstrated by radioactivity measurements on homogenized brain tissue extracts. We infer from these results that [(18)F]5 is a highly promising candidate radiotracer with which to image FAAH in human subjects using PET and clinical studies are proceeding.

Radiosynthesis and evaluation of [¹¹C-carbonyl]-labeled carbamates as fatty acid amide hydrolase radiotracers for positron emission tomography.

Fatty acid amide hydrolase (FAAH) plays a key role in regulating the tone of the endocannabinoid system. Radiotracers are required to image and quantify FAAH activity in vivo. We have synthesized a series of potent FAAH inhibitors encompassing two classes of N-alkyl-O-arylcarbamates and radiolabeled eight of them with carbon-11. The [¹¹C-carbonyl]-radiotracers were evaluated in vitro and ex vivo in rats as potential FAAH imaging agents for positron emission tomography (PET). Both sets of [¹¹C]O-arylcarbamates showed good to excellent brain penetration and an appropriate regional distribution. Pretreatments with a FAAH inhibitor demonstrated that 80-95% of brain uptake of radioactivity constituted binding of the radiotracers to FAAH. Brain extraction measurements showed that binding to FAAH was irreversible and kinetically different for the two classes of carbamates. These promising results are discussed in terms of the requirements of a suitable radiotracer for the in vivo imaging of FAAH using PET.

Development of new carbon-11 labelled radiotracers for imaging GABAA- and GABAB-benzodiazepine receptors.

Two quinolines identified as positive allosteric modulators of γ-aminobutyric acid (GABA)(A) receptors containing the α(2) subunit, 9-amino-2-cyclobutyl-5-(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (4) and 9-amino-2-cyclobutyl-5-(2-methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (5), were radiolabelled at the methoxy position with carbon-11 (half-life=20.4 min). These quinolines represent a new class of potential radiotracers for imaging the benzodiazepine site of GABA(A) receptors with positron emission tomography (PET). Both radiotracers were reliably isolated following reaction of their respective pyridinone/pyridinol tautomeric precursors with [(11)C]CH(3)I in clinically useful, formulated quantities (2.9% and 2.7% uncorrected radiochemical yield, respectively, relative to [(11)C]CO(2)) with high specific activities (>70 GBq µ mol(-1); >2 Ci µ mol(-1)) and high radiochemical purities (>95%). The radiosyntheses reported herein represent rare examples of selectively isolating radiolabelled compounds bearing [(11)C]2-methoxypyridine moieties. Although both radiotracers demonstrated promising imaging characteristics based on preliminary ex vivo biodistribution studies in conscious rodents, higher brain uptake was observed with [(11)C]5 and therefore this radiotracer was further evaluated. Carbon-11 labelled 5 readily penetrated the brain (>1 standard uptake value in cortical regions at 15 min post-injection of the radiotracer), had an appropriate regional brain distribution for GABA(A) receptors that appeared to be reversible, and did not show any appreciable radiometabolites in rat brain homogenates up to 15 min post-injection. Preadministration of flumazenil (1, 10 mg kg(-1)) or 5 (5 mg kg(-1)) effectively blocked >50% of [(11)C]5 binding to the GABA(A) receptor-rich regions, thereby suggesting that this radiotracer is worthy of further evaluation for imaging GABA(A) receptors. Additionally (R,S)-N-(1-(3-chloro-4-methoxyphenyl)ethyl)-3,3-diphenylpropan-1-amine, 6, an allosteric modulator of GABA(B) receptors, was efficiently labelled in one step using [(11)C]methyl iodide. Ex vivo biodistribution studies in conscious rats showed low brain uptake, therefore, efforts are underway to discover alternative radiotracers to image GABA(B). In conclusion, [(11)C]5 is worthy of further evaluation in higher species for imaging GABA(A) receptors in the central nervous system.

Development of new radiopharmaceuticals for imaging monoamine oxidase B.

INTRODUCTION: Imaging monoamine oxidase B (MAO-B) in the central nervous system with PET is an important goal for psychiatric studies. We here report an improved and automated radiosynthesis of N-(6-[(18)F]-fluorohexyl)-N-methylpropargylamine ([(18)F]FHMP; [(18)F]-1), as well as the radiosynthesis of two new promising candidates for imaging cerebral MAO-B, namely, carbon-11-labeled 3-(4-[(11)C]-methoxyphenyl)-6-methyl-2H-1-benzopyran-2-one ([(11)C]-2) and N-((1H-pyrrol-2-yl)methyl)-N-[(11)C]-methyl-1-phenylmethanamine ([(11)C]-3). METHODS: Fluorine-18-labeled 1 was prepared via a tosyloxy precursor in 29%±5% uncorrected radiochemical yield, relative to [(18)F]-fluoride. Both carbon-11-labeled compounds were prepared with [(11)C]CH(3)I using the "LOOP" method in 11% and 18% uncorrected radiochemical yields, respectively, relative to starting [(11)C]CO(2). All radiotracers had specific activities >37 GBq/µmol and were >98% radiochemically pure at end of synthesis (<40 min). All radiotracers were evaluated by ex vivo biodistribution studies in conscious rodents. RESULTS: A major radioactive metabolite in the rodent brain was observed following administration of [(18)F]-1. While [(11)C]-2 had moderate brain penetration and good clearance from normal brain tissue, distribution of radioactivity in brain was indicative of free and nonspecific binding. Good brain uptake was observed with [(11)C]-3 (0.8%-1.4% injected dose per gram at 5 min postinjection), binding appeared to be reversible and distribution conformed with regional distribution of MAO-B in the rat brain. Preinjection of 3 or L-deprenyl showed a modest reduction (up to 25%) of brain activity. CONCLUSION: Carbon-11-labeled 3 was found to have the most favorable properties of the radiotracers evaluated; however, the signal-to-noise ratio was too low to warrant further in vivo imaging studies. Alternative radiotracers for imaging MAO-B are under development.

Monoamine oxidase A inhibitor occupancy during treatment of major depressive episodes with moclobemide or St. John's wort: an [11C]-harmine PET study.

BACKGROUND: Monoamine oxidase A (MAO-A) inhibitor antidepressants raise levels of multiple monoamines, whereas the selective serotonin reuptake inhibitors (SSRIs) only raise extracellular serotonin. Despite this advantage of MAO-A inhibitors, there is much less frequent development of MAO inhibitors compared with SSRIs. We sought to measure brain MAO-A occupancy after 6 weeks of treatment in depressed patients with a clinically effective dose of a selective MAO-A inhibitor and measure MAO-A occupancy after repeated administration of St. John's wort, an herb purported to have MAO-A inhibitor properties. METHODS: Participants underwent 2 [(11)C]-harmine positron emission tomography scans. Healthy controls completed a test-retest condition, and depressed patients were scanned before and after repeated administration of moclobemide or St. John's wort for 6 weeks at the assigned dose. We measured MAO-A VT, an index of MAO-A density, in the prefrontal, anterior cingulate and anterior temporal cortices, putamen, thalamus, midbrain and hippocampus. RESULTS: We included 23 participants (10 controls and 13 patients with major depressive disorder [MDD]) in our study. Monoamine oxidase A VT decreased significantly throughout all regions after moclobemide treatment in patients with MDD compared with controls (repeated-measures analysis of variance, F1,15 = 71.08-130.06, p < 0.001 for all regions, mean occupancy 74% [standard deviation 6%]). Treatment with St. John's wort did not significantly alter MAO-A VT. LIMITATIONS: The occupancy estimates are limited by the sample size of each treatment group; hence, our estimate for the overall moclobemide occupancy of 74% has a 95% confidence interval of 70%-78%, and we can estimate with 95% certainty that the occupancy of St. John's wort is less than 5%. CONCLUSION: For new MAO-A inhibitors, about 74% occupancy at steady-state dosing is desirable. Consistent with this, St. John's wort should not be classified as an MAO-A inhibitor. The magnitude of MAO-A blockade during moclobemide treatment exceeds the elevation of MAO-A binding during illness by at least 30%, suggesting that the treatment effect should exceed the disease effect when designing selective antidepressants for this target.

[11C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography.

INTRODUCTION: Fatty acid amide hydrolase (FAAH) is the enzyme responsible for metabolising the endogenous cannabinoid, anandamide, and thus represents an important target for molecular imaging. To date, no radiotracer has been shown to be useful for imaging of FAAH using either positron emission tomography (PET) or single photon emission computed tomography (SPECT). We here determine the suitability of a novel carbon-11-labeled inhibitor of FAAH via ex vivo biodistribution studies in rat brain in conjunction with pharmacological challenges. METHODS: A potent irreversible inhibitor of FAAH, URB694, radiolabeled with carbon-11 in the carbonyl position ([(11)C]CURB), was administered to male rats via tail-vein injection. Rats were sacrificed at various time points postinjection, and tissue samples were dissected, counted and weighed. Specific binding to FAAH was investigated by pretreatment of animals with URB694 or URB597. For metabolism and mechanism of binding studies, whole brains were excised post-radiotracer injection, homogenised and extracted exhaustively with 80% aq. acetonitrile to determine the time course and fraction of radioactivity that was irreversibly bound to brain parenchyma. RESULTS: Upon intravenous injection into rats, [(11)C]CURB showed high brain uptake [standard uptake value (SUV) of 1.6-2.4 at 5 min] with little washout over time, which is characteristic of irreversible binding. Highest uptake of radioactivity was seen in the cortex, intermediate in the cerebellum and lowest in the hypothalamus, reflecting the reported distribution of FAAH. Brain uptake of radioactivity was decreased in a dose-dependent manner by pretreatment with increasing amounts of URB694, demonstrating that binding was saturable. Pretreatment with the well-characterised FAAH inhibitor, URB597, reduced binding in all brain regions by 70-80%. Homogenised brain extraction experiments demonstrated unequivocally that [(11)C]CURB was irreversibly bound to FAAH. CONCLUSIONS: The title radiotracer demonstrates favourable properties such as good brain uptake, regional heterogeneity and specificity of binding based on ex vivo biodistribution studies in conscious rat brain. [(11)C]CURB represents a highly promising radiotracer for the imaging of FAAH using PET.

Systemic catechol-O-methyl transferase inhibition enables the D1 agonist radiotracer R-[11C]SKF 82957.

INTRODUCTION: R-[(11)C]-SKF 82957 is a high-affinity and potent dopamine D(1) receptor agonist radioligand, which gives rise to a brain-penetrant lipophilic metabolite. In this study, we demonstrate that systemic administration of catechol-O-methyl transferase (COMT) inhibitors blocks this metabolic pathway, facilitating the use of R-[(11)C]-SKF 82957 to image the high-affinity state of the dopamine D(1) receptor with PET. METHODS: R-[(11)C]SKF 82957 was administered to untreated and COMT inhibitor-treated conscious rats, and the radioactive metabolites present in the brain and plasma were quantified by HPLC. Under optimal conditions, cerebral uptake and dopamine D(1) binding of R-[(11)C]SKF 82957 were measured ex vivo. In addition, pharmacological challenges with the receptor antagonist SCH 23390, amphetamine, the dopamine reuptake inhibitor RTI-32 and the dopamine hydroxylase inhibitor α-methyl-p-tyrosine were performed to study the specificity and sensitivity of R-[(11)C]-SKF 82957 dopamine D(1) binding in COMT-inhibited animals. RESULTS: Treatment with the COMT inhibitor tolcapone was associated with a dose-dependent (EC(90) 5.3 ± 4.3 mg/kg) reduction in the lipophilic metabolite. Tolcapone treatment (20 mg/kg) also resulted in a significant increase in the striatum/cerebellum ratio of R-[(11)C]SKF 82957, from 15 (controls) to 24. Treatment with the dopamine D(1) antagonist SCH 23390 reduced the striatal binding to the levels of the cerebellum, demonstrating a high specificity and selectivity of R-[(11)C]SKF 82957 binding. CONCLUSIONS: Pre-treatment with the COMT inhibitor tolcapone inhibits formation of an interfering metabolite of R-[(11)C]SKF 82957. Under such conditions, R-[(11)C]SKF 82957 demonstrates high potential as the first agonist radiotracer for imaging the dopamine D(1) receptor by PET.

Radiosynthesis and initial evaluation of [18F]-FEPPA for PET imaging of peripheral benzodiazepine receptors.

INTRODUCTION: A novel [18F]-radiolabelled phenoxyanilide, [18F]-FEPPA, has been synthesized and evaluated, in vitro and ex vivo, as a potential positron emission tomography imaging agent for the peripheral benzodiazepine receptor (PBR). METHODS: [18F]-FEPPA and two other radiotracers for imaging PBR, namely [11C]-PBR28 and [11C]-PBR28-d3, were synthesised and evaluated in vitro and ex vivo as potential PBR imaging agents. RESULTS: [18F]-FEPPA is efficiently prepared in one step from its tosylate precursor and [18F]-fluoride in high radiochemical yields and at high specific activity. FEPPA displayed a Ki of 0.07 nM for PBR in rat mitochondrial membrane preparations and a suitable lipophilicity for brain penetration (log P of 2.99 at pH 7.4). Upon intravenous injection into rats, [18F]-FEPPA showed moderate brain uptake [standard uptake value (SUV) of 0.6 at 5 min] and a slow washout (SUV of 0.35 after 60 min). Highest uptake of radioactivity was seen in the hypothalamus and olfactory bulb, regions previously reported to be enriched in PBR in rat brain. Analysis of plasma and brain extracts demonstrated that [18F]-FEPPA was rapidly metabolized, but no lipophilic metabolites were observed in either preparation and only 5% radioactive metabolites were present in brain tissue extracts. Blocking studies to determine the extent of specific binding of [18F]-FEPPA in rat brain were problematic due to large perturbations in circulating radiotracer and the lack of a reference region. CONCLUSIONS: Further evaluation of the potential of [18F]-FEPPA will require the employment of rigorous kinetic models and/or appropriate animal models.

Opposite effects of sleep rebound on orexin OX1 and OX2 receptor expression in rat brain.

Orexins (hypocretins) have been implicated in the regulation of the normal sleep-wake cycle, in sensorimotor programming, and in other homeostatic and neuroregulatory processes. The present study examined the effects of sleep deprivation (SD) and sleep recovery on the expression of orexin 1 receptors (OX1R) and orexin 2 receptors (OX2R) throughout the brain. Rats were sacrificed either immediately after 96 h of sleep deprivation (SD group) or after SD followed by 24 h of sleep recovery (Rebound group). Prepro-orexin mRNA showed a non-significant increase in the SD group relative to controls, but a pronounced and significant increase in the Rebound group (+88%, P < 0.007). Similarly, sleep deprivation produced no effect on OX1R or OX2R mRNA levels. However, in the Rebound group, OX1R mRNA levels increased significantly, compared to either control or SD groups, in 37 of 92 brain regions analyzed, with particularly strong effects in the amygdala and hypothalamus. Changes in OX2R mRNA levels were also seen only in the sleep Rebound group, but they were fewer in number (10 out of 86 regions), were in the direction of decreased rather than increased expression, and were predominantly confined to cerebral cortical areas. These observations indicate that some factor associated with sleep recovery, possibly the compensatory increase in REM sleep, has strong effects on the orexin system at the mRNA level. They further indicate that,pOX1 and OX2 receptors are affected in opposite way and that the former are more vulnerable to these effects than the latter.

Evaluation of the motor initiation hypothesis of APD-induced conditioned avoidance decreases.

Antipsychotic drugs (APDs) selectively disrupt conditioned avoidance responding (CAR)--a feature that distinguishes them from all other psychotropics. It is thought that this effect reflects their effect on motor initiation; however, this conclusion is questionable because most studies it relies on have often examined avoidance responding under APD treatment, and tested animals with preshock stimuli followed by the footshock. APD-induced CAR effects are confounded by APDs' motor effects and by the presence of footshock. The objective of this study was to evaluate the motor initiation hypothesis by testing animals without drug and under extinction conditions. In Experiment 1, we administered haloperidol, clozapine or chlordiazepoxide (an anxiolytic as a pharmacological control) during the acquisition phase of CAR, but tested animals 2 days later. The APD-induced CAR disruption was present even in the absence of the drug and footshock. In Experiment 2, we first trained rats to a learning criterion, and then subjected them to 4 days of CAR extinction training under drug or vehicle. In the subsequent CAR extinction tests, the rats previously treated with APDs still showed significantly lower avoidance responses. In both experiments, the effects of haloperidol and clozapine were distinct from those of chlordiazepoxide. These data suggest that APD-induced CAR decreases cannot be explained as the unconditioned motor impairment effects of APDs, but probably reflect a dopamine-blockade-mediated change in incentive motivation.