Summary The Molecular Imaging Branch (MIB) aims to exploit positron emission tomography (PET) as an imaging technique for investigating neuropsychiatric disorders, such as autism, depression, addiction, schizophrenia, and Alzheimer's Disease. Fundamental to this mission is the development of novel radioactive probes (radiotracers) that can be used with PET to measure changes in low level proteins in the brains of living human subjects where these proteins are suspected to have critical involvement in the progression of neuropsychiatric disorders. Such proteins include some neuroreceptors, transporters, enzymes, and plaques. PET is uniquely powerful and sensitive when coupled with the use of biochemically-specific PET radiotracers. The chemical development of new radiotracers is the key to exploiting the full potential of PET in neuropsychiatric research. However, a successful PET radiotracer must satisfy a wide range of difficult-to-satisfy chemical, biochemical, and pharmacological criteria. Consequently, PET radiotracer development is highly challenging. In fact, our research has some parallels with drug discovery in that it entails high effort and heavy risk but can reap rich biomedical rewards. As of now, the number of potentially interesting PET imaging targets (brain proteins) far exceeds the range of currently available and useful radiotracers. Within MIB, the PET Radiopharmaceutical Sciences Section (PRSS) places a concerted effort on all chemical aspects of PET radiotracer discovery. Our laboratories are equipped for medicinal chemistry and automated radiochemistry with positron-emitting carbon-11 (t1/2 = 20 min) and fluorine-18 (t1/2 = 110 min). These two very short-lived radioisotopes are available to us daily from the adjacent cyclotrons of the NIH Clinical Center (Chief: Dr. P. Herscovitch). Our Section interacts seamlessly with the Section on PET Neuroimaging Sciences in our Branch (Chief: Dr. R.B. Innis) for early evaluation of candidate radiotracers in biological models and in animals. Subsequent PET research in humans is also performed in collaboration with the Imaging Section under Food and Drug Administration oversight through 'exploratory' or 'full' Investigational New Drug applications. All radiotracer production for PET studies in humans is now performed within the state-of-the-art NIH Clinical Center current good manufacturing practice (CGMP) laboratory. In the period covered by this report, we worked on developing and producing PET radiotracers for several protein targets. These include translocator protein 18 kDa (TSPO); the NR2B sub-site of the NMDA receptor, serotonin subtype 1B (5-HT1B)receptors, and several enzymes (COX-1, COX-2, PDE1 and PDE4 subtypes, and OGA). One radiotracer that we earlier developed for TSPO imaging (C-11PBR28) has been applied by many imaging centers to investigate brain inflammatory conditions (i.e., neuroinflammation) in response to various neurological insults (e.g., stroke, epilepsy and neurodegeneration). An unexpected finding is that healthy human subjects, because of a genetic difference, carry either one or both of two distinct forms of TSPO and that these interact differently with C-11PBR28, complicating the analysis of PET studies. Consequently, we sought to develop genetically-insensitive TSPO radiotracers. We explored new chemotypes with potential to provide superior PET radiotracers for TSPO. One of our new radiotracers, C-11ER176, appeared promising for avoiding genotype sensitivity based on our evaluation in animals and in human tissue in vitro. In addition, while C-11ER176 does not show the expected genotype insensitivity in living humans, it does turn out to be one of the highest performing TSPO radiotracers yet known, and especially is able to quantify TSPO in all subjects of identified genotype. We are now producing this radiotracer for clinical studies in human subjects and other imaging centers plan to do so for this purpose. We are also developing longer-lived and therefore more broadly useful F-18 labeled versions of this radiotracer. Some of these already show high promise for continued development. We have been developing radiotracers for other targets relevant to the study of neuroinflammation, such as the cyclooxygenase (COX) subtype 1 and subtype 2 enzymes. Very promising C-11 labeled radiotracers have been identified, and two of these have now been evaluated in human subjects, and will soon enter application in clinical studies. These radiotracers may provide more biochemical and cellular specificity for investigation of neuroinflammation. They can also prove useful for the development of improved anti-inflammatory drugs. N-Methyl-D-aspartate (NMDA) receptors are proteins which are acted upon by glutamate for implementing normal brain function. Perturbations in NMDA function are strongly implicated in the pathogenesis of schizophrenia and other neuropsychiatric disorders, such as depression. Our research has led to promising radiotracers for imaging the NR2B binding site on the NMDA receptor in rodents and non-human primate. We are planning to evaluate one of these in human subjects. PET radiotracers can provide important quantitative information on experimental therapeutics for neuropsychiatric disorders, such as ability to cross the blood-brain-barrier and to engage with a target protein. In collaborations with academia and Pharma, we are developing several radiotracers for this purpose. These radiotracers are targeted at proteins that have not previously been imaged in living human brain that may have eventual clinical research utility. These proteins include, with clinical interest in parentheses, the enzyme OGA (dementia) and subtypes of phosphodiesterase (depression, cognitive function). Our radiotracer for imaging OGA has been found to perform excellently in human subjects. The development of radiotracers for phosphodiesterase subtypes has shown some promise. One radiotracer performed acceptably in non-human primate but less well in human. Alternatives with improved properties are now being in development. Our development of NR2B radioligands has attracted attention and collaboration from Pharma for the possibility to develop new antidepressants. We are advancing methodology for improved radiotracer development. Thus, we recently developed new agents for radiolabeling, notably C-11fluoroform and 18Ffluoroform. These expand chemical space for radiotracer development. We continue developing radiolabeling chemistries with these agents for preparing new radiotracers. For example, we have recently produced new TSPO radiotracers through these means. New methods for utilizing cyclotron-produced F-18fluoride ion and C-11 carbon monoxide are being developed. A sensitive mass spectrometry method for measuring blood radiotracer concentration following intravenous administration has been developed and evaluated. Such measurements are required to analyze PET experiments quantitatively. The mass spectrometry method avoids the demanding logistics associated with measuring fast-decaying radioactivity. Productive collaborations continue with external academic chemistry and medicinal chemistry laboratories, nationally and internationally, and with Pharma. Productive collaborations also exist with other PET centers on radiochemistry and radiotracer development. The laboratory is active in training new scientists for this field at graduate and postdoctoral level. We produce some useful radiotracers that have been developed elsewhere for PET investigations in animal or human subjects e.g., C-11rolipram (for PDE4 enzyme imaging), and C-11-labeled radiotracers for investigation of DREADD technology. Each PET experiment requires a radiosynthesis of the radiotracer on the same day, and hence radiotracer production is a regular activity.