Radiosynthesis and evaluation of [¹¹C-carbonyl]-labeled carbamates as fatty acid amide hydrolase radiotracers for positron emission tomography.
ABSTRACT: 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.
Project description:Positron emission tomography (PET) is a molecular imaging modality that enables in vivo exploration of metabolic processes and especially the pharmacology of neuroreceptors. G protein-coupled receptors (GPCRs) play an important role in numerous pathophysiologic disorders of the central nervous system. Thus, they are targets of choice in PET imaging to bring proof concept of change in density in pathological conditions or in pharmacological challenge. At present, most radiotracers are antagonist ligands. In vitro data suggest that properties differ between GPCR agonists and antagonists: antagonists bind to receptors with a single affinity, whereas agonists are characterized by two different affinities: high affinity for receptors that undergo functional coupling to G-proteins, and low affinity for those that are not coupled. In this context, agonist radiotracers may be useful tools to give functional images of GPCRs in the brain, with high sensitivity to neurotransmitter release. Here, we review all existing PET radiotracers used from animals to humans and their role for understanding the ligand-receptor paradigm of GPCR in comparison with corresponding antagonist radiotracers.
Project description:Positron emission tomography (PET) is an in vivo molecular imaging tool which is widely used in nuclear medicine for early diagnosis and treatment follow-up of many brain diseases. PET uses biomolecules as probes which are labeled with radionuclides of short half-lives, synthesized prior to the imaging studies. These probes are called radiotracers. Fluorine-18 is a radionuclide routinely used in the radiolabeling of neuroreceptor ligands for PET because of its favorable half-life of 109.8 min. The delivery of such radiotracers into the brain provides images of transport, metabolic, and neurotransmission processes on the molecular level. After a short introduction into the principles of PET, this review mainly focuses on the strategy of radiotracer development bridging from basic science to biomedical application. Successful radiotracer design as described here provides molecular probes which not only are useful for imaging of human brain diseases, but also allow molecular neuroreceptor imaging studies in various small-animal models of disease, including genetically-engineered animals. Furthermore, they provide a powerful tool for in vivo pharmacology during the process of pre-clinical drug development to identify new drug targets, to investigate pathophysiology, to discover potential drug candidates, and to evaluate the pharmacokinetics and pharmacodynamics of drugs in vivo.
Project description:The two major endocannabinoid transmitters, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are degraded by distinct enzymes in the nervous system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL inhibitors cause elevations in brain AEA and 2-AG levels, respectively, and reduce pain, anxiety, and depression in rodents without causing the full spectrum of psychotropic behavioral effects observed with direct cannabinoid receptor-1 (CB1) agonists. These findings have inspired the development of several classes of endocannabinoid hydrolase inhibitors, most of which have been optimized to show specificity for either FAAH or MAGL or, in certain cases, equipotent activity for both enzymes. Here, we investigate an unusual class of O-hydroxyacetamide carbamate inhibitors and find that individual compounds from this class can serve as selective FAAH or dual FAAH/MAGL inhibitors in vivo across a dose range (0.125-12.5 mg kg(-1)) suitable for behavioral studies. Competitive and click chemistry activity-based protein profiling confirmed that the O-hydroxyacetamide carbamate SA-57 is remarkably selective for FAAH and MAGL in vivo, targeting only one other enzyme in brain, the additional 2-AG hydrolase ABHD6. These data designate O-hydroxyacetamide carbamates as a versatile chemotype for creating endocannabinoid hydrolase inhibitors that display excellent in vivo activity and tunable selectivity for FAAH-anandamide versus MAGL (and ABHD6)-2-AG pathways.
Project description:BACKGROUND:Serotonin 1A (5-HT1A) receptors are implicated in the pathogenesis of several psychiatric and neurodegenerative disorders motivating the development of suitable radiotracers for in vivo positron emission tomography (PET) neuroimaging. The gold standard PET imaging agent for this target is [carbonyl-11C]WAY-100635, labeled via a technically challenging multi-step reaction that has limited its widespread use. While several antagonist and agonist-based PET radiotracers for 5-HT 1A receptors have been developed, their clinical translation has been hindered by methodological challenges and/or and non-specific binding. As a result, there is continued interest in the development of new and more selective 5-HT1A PET tracers having a relatively easier and reliable radiosynthesis process for routine production and with favorable metabolism to facilitate tracer-kinetic modeling. The purpose of the current study was to develop and characterize a radioligand with suitable characteristics for imaging 5-HT1A receptors in the brain. The current study reports the in vitro characterization and radiosyntheses of three candidate 5-HT1A receptor antagonists, DF-100 (1), DF-300 (2) and DF-400 (3), to explore their suitability as potential PET radiotracers. RESULTS:Syntheses of 1-3 and corresponding precursors for radiolabeling were achieved from isonicotinic, picolinic acid or picolino nitrile. In vitro binding studies demonstrated nanomolar affinity of the compounds for 5-HT1A receptors. Binding of 1-3 for other biogenic amines, neurotransmitter receptors, and transporters was negligible with the exception of moderate affinities for ?1-adrenergic receptors (4-6-fold less potent than that for 5-HT1A receptor). Radioligands [11C]1-3 were efficiently prepared by 11C-O-methylation of the corresponding phenolic precursor in non-decay corrected radiochemical yields of 7-11% with >?99% chemical and radiochemical purities. Dynamic PET studies in rats demonstrated negligible brain uptake of [11C]1 and [11C]2. In contrast, significant brain uptake of [11C]3 was observed with an early peak SUV of 4-5. However, [11C]3 displayed significant off-target binding attributed to ?1-adrenergic receptors based on regional distribution (thalamus>hippocampus) and blocking studies. CONCLUSION:Despite efficient radiolabeling, results from PET imaging experiments limit the application of [11C]3 for in vivo quantification of 5-HT1A receptors. Nevertheless, derivatives of compound 3 may provide a scaffold for alternative PET radiotracers with improved selectivity for 5-HT 1A receptors or ?1-adrenergic receptors.
Project description:The advent of preclinical research scanners for in vivo imaging of small animals has added confidence into the multi-step decision-making process of radiotracer discovery and development. Furthermore, it has expanded the utility of imaging techniques available to dissect clinical questions, fostering a cyclic interaction between the clinical and the preclinical worlds. Significant efforts from medicinal chemistry have also made available several high-affinity and selective compounds amenable for radiolabeling, that target different receptors, transporters and enzymes in vivo. This substantially increased the range of applications of molecular imaging using positron emission tomography (PET) or single photon emission computed tomography (SPECT). However, the process of developing novel radiotracers for in vivo imaging of the human brain is a multi-step process that has several inherent pitfalls and technical difficulties, which often hampers the successful translation of novel imaging agents from preclinical research into clinical use. In this paper, the process of radiotracer development and its relevance in brain research is discussed; as well as, its pitfalls, technical challenges and future promises. Examples of successful and unsuccessful translation of brain radiotracers will be presented.
Project description:Antibodies, and engineered antibody fragments, labeled with radioisotopes are being developed as radiotracers for the detection and phenotyping of diseases such as cancer. The development of antibody-based radiotracers requires extensive characterization of their in vitro and in vivo properties, including their ability to target tumors in an antigen-selective manner. In this study, we investigated the use of Cerenkov luminescence imaging (CLI) as compared with PET as a modality for evaluating the in vivo behavior of antibody-based radiotracers.The anti-prostate-specific membrane antigen (PSMA) huJ591 antibody (IgG; 150 kDa) and its minibody (Mb; 80 kDa) format were functionalized with the chelator 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) and radiolabeled with the positron-emitting radionuclide 64Cu (half-life, 12.7 h). Immunoreactive preparations of the radiolabeled antibodies were injected into NCr nu/nu mice harboring PSMA-positive CWR22Rv1 and PSMA-negative PC-3 tumor xenografts. Tumor targeting was evaluated by both PET and CLI.64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb retained the ability to bind cell surface PSMA, and both radiotracers exhibited selective uptake into PSMA-positive tumors. Under the experimental conditions used, PSMA-selective uptake of 64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb was observed by CLI as early as 3 h after injection, with tumor-to-background ratios peaking at 24 (IgG) and 16 (Mb) h after injection. Targeting data generated by CLI correlated with that generated by PET and necropsy.CLI provided a rapid and simple assessment of the targeting specificity and pharmacokinetics of the antibody-based PET radiotracers that correlated well with the behavior observed by standard PET imaging. Moreover, CLI provided clear discrimination between uptake kinetics of an intact IgG and its small-molecular-weight derivative Mb. These data support the use of CLI for the evaluation of radiotracer performance.
Project description:Fatty acid amide hydrolase (FAAH) is one of the principle enzymes for metabolizing endogenous cannabinoid neurotransmitters such as anandamide, and thus regulates endocannabinoid (eCB) signaling. Selective pharmacological blockade of FAAH has emerged as a potential therapy to discern the endogenous functions of anandamide-mediated eCB pathways in anxiety, pain, and addiction. Quantification of FAAH in the living brain by positron emission tomography (PET) would help our understanding of the endocannabinoid system in these conditions. While most FAAH radiotracers operate by an irreversible ("suicide") binding mechanism, a FAAH tracer with reversibility would facilitate quantitative analysis. We have identified and radiolabeled a reversible FAAH inhibitor, 7-(2-[(11)C]methoxyphenyl)-1-(5-(pyridin-2-yl)oxazol-2-yl)heptan-1-one ([(11)C]MPPO) in 13% radiochemical yield (nondecay corrected) with >99% radiochemical purity and 2 Ci/?mol (74 GBq/?mol) specific activity. The tracer showed moderate brain uptake (0.8 SUV) with heterogeneous brain distribution. However, blocking studies with a potent FAAH inhibitor URB597 demonstrated a low to modest specificity to the target. Measurement of lipophilicity, metabolite, and efflux pathway analysis were also performed to study the pharmacokinetic profile of [(11)C]MPPO. In all, we reported an efficient radiolabeling and preliminary evaluation of the first-in-class FAAH inhibitor [(11)C]MPPO with ?-ketoheterocyclic scaffold.
Project description:Standardized reporting is more and more routinely implemented in clinical practice, and such structured reports have a major impact on a large variety of medical fields, such as laboratory medicine, pathology, and, recently, radiology. Notably, the field of nuclear medicine is constantly evolving as novel radiotracers for numerous clinical applications are developed. Thus, framework systems for standardized reporting in this field may increase clinical acceptance of new radiotracers, allow for inter- and intracenter comparisons for quality assurance, and be used in global multicenter studies to ensure comparable results and enable efficient data abstraction. In the last couple of years, several standardized framework systems for PET radiotracers with potential theranostic applications have been proposed. These include systems for prostate-specific membrane antigen-targeted PET agents to diagnose and treat prostate cancer, and systems for somatostatin receptor-targeted PET agents to diagnose and treat neuroendocrine neoplasia. In the present review, the framework systems for these 2 types of cancer will be briefly introduced, followed by an overview of their advantages and limitations. In addition, potential applications will be defined, approaches to validate such concepts will be proposed, and future perspectives will be discussed.
Project description:Dosimetry models using preclinical positron emission tomography (PET) data are commonly employed to predict the clinical radiological safety of novel radiotracers. However, unbiased clinical safety profiling remains difficult during the translational exercise from preclinical research to first-in-human studies for novel PET radiotracers. In this study, we assessed PET dosimetry data of six 18F-labelled radiotracers using preclinical dosimetry models, different reconstruction methods and quantified the biases of these predictions relative to measured clinical doses to ease translation of new PET radiotracers to first-in-human studies. Whole-body PET images were taken from rats over 240 min after intravenous radiotracer bolus injection. Four existing and two novel PET radiotracers were investigated: [18F]FDG, [18F]AlF-NOTA-RGDfK, [18F]AlF-NOTA-octreotide ([18F]AlF-NOTA-OC), [18F]AlF-NOTA-NOC, [18F]ENC2015 and [18F]ENC2018. Filtered-back projection (FBP) and iterative methods were used for reconstruction of PET data. Predicted and true clinical absorbed doses for [18F]FDG and [18F]AlF-NOTA-OC were then used to quantify bias of preclinical model predictions versus clinical measurements. Our results show that most dosimetry models were biased in their predicted clinical dosimetry compared to empirical values. Therefore, normalization of rat:human organ sizes and correction for reconstruction method biases are required to achieve higher precision of dosimetry estimates.
Project description:BACKGROUND:In the US, EU and elsewhere, basic clinical research studies with positron emission tomography (PET) radiotracers that are generally recognized as safe and effective (GRASE) can often be conducted under institutional approval. For example, in the United States, such research is conducted under the oversight of a Radioactive Drug Research Committee (RDRC) as long as certain requirements are met. Firstly, the research must be for basic science and cannot be intended for immediate therapeutic or diagnostic purposes, or to determine the safety and effectiveness of the PET radiotracer. Secondly, the PET radiotracer must be generally recognized as safe and effective. Specifically, the mass dose to be administered must not cause any clinically detectable pharmacological effect in humans, and the radiation dose to be administered must be the smallest dose practical to perform the study and not exceed regulatory dose limits within a 1-year period. In our experience, the main barrier to using a PET radiotracer under RDRC approval is accessing the required information about mass and radioactive dosing. RESULTS:The University of Michigan (UM) has a long history of using PET radiotracers in clinical research studies. Herein we provide dosing information for 55 radiotracers that will enable other PET Centers to use them under the approval of their own RDRC committees. CONCLUSIONS:The data provided herein will streamline future RDRC approval, and facilitate further basic science investigation of 55 PET radiotracers that target functionally relevant biomarkers in high impact disease states.