Michelle L. James

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

Molecular imaging is revolutionizing the way we study the inner workings of the human body, diagnose diseases, approach drug design, and assess therapies. The field as a whole is making possible the visualization of complex biochemical processes involved in normal physiology and disease states, in real time, in living cells, tissues, and intact subjects. In this review, we focus specifically on molecular imaging of intact living subjects. We provide a basic primer for those who are new to molecular imaging, and a resource for those involved in the field. We begin by describing classical molecular imaging techniques together with their key strengths and limitations, after which we introduce some of the latest emerging imaging modalities. We provide an overview of the main classes of molecular imaging agents (i.e., small molecules, peptides, aptamers, engineered proteins, and nanoparticles) and cite examples of how molecular imaging is being applied in oncology, neuroscience, cardiology, gene therapy, cell tracking, and theranostics (therapy combined with diagnostics). A step-by-step guide to answering biological and/or clinical questions using the tools of molecular imaging is also provided. We conclude by discussing the grand challenges of the field, its future directions, and enormous potential for further impacting how we approach research and medicine.

Comparative Evaluation of the Translocator Protein Radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a Rat Model of Acute Neuroinflammation

Overexpression of the translocator protein, TSPO (18 kDa), formerly known as the peripheral benzodiazepine receptor, is a hallmark of activation of cells of monocytic lineage (microglia and macrophages) during neuroinflammation. Radiolabeling of TSPO ligands enables the detection of neuroinflammatory lesions by PET. Two new radioligands, 11C-labeled N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-yl]acetamide (DPA-713) and 18F-labeled N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-yl)acetamide (DPA-714), both belonging to the pyrazolopyrimidine class, were compared in vivo and in vitro using a rodent model of neuroinflammation. Methods: 11C-DPA-713 and 18F-DPA-714, as well as the classic radioligand 11C-labeled (R)-N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide (PK11195), were used in the same rat model, in which intrastriatal injection of (R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique gave rise to a strong neuroinflammatory response. Comparative endpoints included in vitro autoradiography and in vivo imaging on a dedicated small-animal PET scanner under identical conditions. Results: 11C-DPA-713 and 18F-DPA-714 could specifically localize the neuroinflammatory site with a similar signal-to-noise ratio in vitro. In vivo, 18F-DPA-714 performed better than 11C-DPA-713 and 11C-PK11195, with the highest ratio of ipsilateral to contralateral uptake and the highest binding potential. Conclusion: 18F-DPA-714 appears to be an attractive alternative to 11C-PK11195 because of its increased bioavailability in brain tissue and its reduced nonspecific binding. Moreover, its labeling with 18F, the preferred PET isotope for radiopharmaceutical chemistry, favors its dissemination and wide clinical use. 18F-DPA-714 will be further evaluated in longitudinal studies of neuroinflammatory conditions such as are encountered in stroke or neurodegenerative diseases.

DPA-714, a New Translocator Protein–Specific Ligand: Synthesis, Radiofluorination, and Pharmacologic Characterization

The translocator protein (18 kDa) (TSPO), formerly known as the peripheral benzodiazepine receptor, is dramatically upregulated under pathologic conditions. Activated microglia are the main cell type expressing the TSPO at sites of central nervous system pathology. Radioligands for the TSPO can therefore measure active disease in the brain. This article details the synthesis, radiofluorination, and pharmacologic evaluation of a new TSPO-specific pyrazolopyrimidine, DPA-714. Methods: The affinity of DPA-714 for the TSPO was measured in rat kidney membranes with 3H-PK11195. The in vitro functional activity of DPA-714 was measured in a steroidogenic assay in which the ability of DPA-714 to increase pregnenolone synthesis was measured with rat C6 glioma cells. The radiofluorination of DPA-714 was achieved by nucleophilic 18F-fluoride displacement of the tosylate precursor. 18F-DPA-714 was assessed in rats harboring unilateral quinolinic acid (QA) lesions. In addition, pretreatment experiments were performed with PK11195 (5 mg/kg), DPA-714 (1 mg/kg), and DPA-713 (1 mg/kg). The in vivo binding and biodistribution of 18F-DPA-714 were determined in a baboon with PET. Experiments involving presaturation with PK11195 (1.5 mg/kg) and displacement with DPA-714 (1 mg/kg) were conducted to evaluate the specificity of radioligand binding. Results: In vitro binding studies revealed that DPA-714 displayed a high affinity for the TSPO (dissociation constant, 7.0 nM). DPA-714 stimulated pregnenolone synthesis at levels 80% above the baseline. 18F-DPA-714 was prepared at a 16% radiochemical yield and a specific activity of 270 GBq/μmol. In rats harboring unilateral QA lesions, an 8-fold-higher level of uptake of 18F-DPA-714 was observed in the ipsilateral striatum than in the contralateral striatum. Uptake in the ipsilateral striatum was shown to be selective because it was inhibited to the level in the contralateral striatum in the presence of PK11195, nonlabeled DPA-714, or DPA-713. PET studies demonstrated rapid penetration and good retention of 18F-DPA-714 in the baboon brain. Pretreatment with PK11195 effectively inhibited the uptake of 18F-DPA-714 in the whole brain, indicating its selective binding to the TSPO. The injection of nonlabeled DPA-714 20 min after the injection of 18F-DPA-714 resulted in radioligand washout, demonstrating the reversibility of 18F-DPA-714 binding. Conclusion: 18F-DPA-714 is a specific radioligand for the TSPO, displaying promising in vivo properties and thus warranting further investigation.

11C-DPA-713: a novel peripheral benzodiazepine receptor PET ligand for in vivo imaging of neuroinflammation.

The induction of neuroinflammatory processes, characterized by upregulation of the peripheral benzodiazepine receptor (PBR) expressed by microglial cells, is well correlated with neurodegenerative diseases and with acute neuronal loss. The continually increasing incidence of neurodegenerative diseases in developed countries has become a major health problem, for which the development of diagnostic and follow-up tools is required. Here we investigated a new PBR ligand suitable for PET to monitor neuroinflammatory processes as an indirect hallmark of neurodegeneration. Methods: We compared PK11195, the reference compound for PBR binding sites, with the new ligand DPA-713 (N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide), using a small-animal dedicated PET camera in a model of neuroinflammation in rats. Seven days after intrastriatal injection of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), a PET scan was performed using 11C-PK11195 or 11C-DPA-713. Immunohistochemistry for neuronal (NeuN), astrocyte (glial fibrillary acidic protein), and microglial (CD11) specific markers as well as 3H-PK11195 autoradiographic studies were then correlated with the imaging data. Results: Seven days after a unilateral injection of AMPA in the striatum, 11C-DPA-713 exhibits a better contrast between healthy and damaged brain parenchyma than 11C-PK11195 (2.5-fold ± 0.14 increase vs. 1.6-fold ± 0.05 increase, respectively). 11C-DPA-713 and 11C-PK11195 exhibit similar brain uptake in the ipsilateral side, whereas, in the contralateral side, 11C-DPA-713 uptake was significantly lower than 11C-PK11195. Modeling of the data using the simplified reference tissue model shows that the binding potential was significantly higher for 11C-DPA-713 than for 11C-PK11195. Conclusion: 11C-DPA-713 displays a higher signal-to-noise ratio than 11C-PK11195 because of a lower level of unspecific binding that is likely related to the lower lipophilicity of 11C-DPA-713. Although further studies in humans are required, 11C-DPA-713 represents a suitable alternative to 11C-PK11195 for PET of PBR as a tracer of neuroinflammatory processes induced by neuronal stress.

Initial Evaluation of 11C-DPA-713, a Novel TSPO PET Ligand, in Humans

Translocator protein (TSPO) is upregulated in activated microglia and thus can serve as a marker of neuroinflammation. Recently, a novel radioligand, 11C-N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide (11C-DPA-713), has been described that binds to TSPO with high affinity. Here, we report the first examination of 11C-DPA-713 in human subjects using PET. Methods: Five healthy controls were studied with PET for 90 min after a bolus injection of high-specific-activity 11C-DPA-713. For comparison, 2 additional healthy controls were studied with 11C-R-PK11195. Arterial blood sampling and metabolite analysis were performed to allow the accurate quantification of tracer kinetics. Tracer uptake was evaluated for several brain regions. Tissue time–activity curves were fitted using 1- and 2-tissue-compartment models, with goodness-of-fit tests showing a preference for the 2-tissue model. Results: In the healthy brain, the average plasma-to-tissue clearance and the total volume of distribution were an order of magnitude larger than measured for 11C-R-PK11195. Accordingly, dose-normalized time–activity curves showed that 11C-DPA-713 gives a larger brain signal. Conclusion: Studies in patient populations will help determine whether 11C-DPA-713 provides better sensitivity for evaluating increased TSPO expression. This initial study in humans shows that 11C-DPA-713 is a promising ligand for evaluating TSPO binding with PET.

Colony-stimulating factor 1 receptor (CSF1R) signaling in injured neurons facilitates protection and survival

Colony-stimulating factor 1 and IL-34 protect against and partially reverse neurodegeneration in mice in part via promoting CREB signaling.

Development of Ligands for the Peripheral Benzodiazepine Receptor

The peripheral benzodiazepine receptor (PBR) initially characterised as a high affinity binding site for diazepam, is densely distributed in most peripheral organs whilst only moderately expressed in the healthy brain. The predominant cell type expressing the PBR at regions of central nervous system (CNS) pathology are activated microglial cells. Under neuroinflammatory conditions there is an over-expression of PBR binding sites indicating that measurements of PBR density can act as a useful index of brain disease activity. The PBR is now considered a significant therapeutic and diagnostic target which has provided the impetus for PBR ligand development. There are several classes of PBR ligands available including benzodiazepines (Ro5-4864), isoquinoline carboxamides (PK 11195), indoleacetamides (FGIN-1-27), phenoxyphenyl-acetamides (DAA1106) and pyrazolopyrimidines (DPA-713). Subsequent conformationally restrained isoquinoline and indoleacetamide analogues have been synthesised in an attempt to yield PBR ligands with superior affinity and brain kinetics. Even though the PBR has been linked to a number of biochemical processes, including cell proliferation, apoptosis, steroidogenesis, porphyrin transport and immunomodulation, its exact physiological role is yet to be deciphered. Selective PBR ligands with favourable in vivo binding properties and kinetics is required to gain a more complete understanding on the normal functioning of the PBR and the chemical pathways underlying several pathological conditions. Novel PBR ligands with unique binding properties and functional activity may also generate information on the localisation of the PBR and the possibility of PBR subtypes. This review highlights the main classes of PBR ligands to date. In addition the biological activity and therapeutic potential of certain PBR ligands is discussed.

Preclinical Evaluation of Raman Nanoparticle Biodistribution for their Potential Use in Clinical Endoscopy Imaging

Raman imaging offers unsurpassed sensitivity and multiplexing capabilities. However, its limited depth of light penetration makes direct clinical translation challenging. Therefore, a more suitable way to harness its attributes in a clinical setting would be to couple Raman spectroscopy with endoscopy. The use of an accessory Raman endoscope in conjunction with topically administered tumor-targeting Raman nanoparticles during a routine colonoscopy could offer a new way to sensitively detect dysplastic lesions while circumventing Ramans limited depth of penetration and avoiding systemic toxicity. In this study, the natural biodistribution of gold surface-enhanced Raman scattering (SERS) nanoparticles is evaluated by radiolabeling them with (64) Cu and imaging their localization over time using micropositron emission tomography (PET). Mice are injected either intravenously (IV) or intrarectally (IR) with approximately 100 microcuries (μCi) (3.7 megabecquerel (MBq)) of (64) Cu-SERS nanoparticles and imaged with microPET at various time points post injection. Quantitative biodistribution data are obtained as % injected dose per gram (%ID g(-1)) from each organ, and the results correlate well with the corresponding microPET images, revealing that IV-injected mice have significantly higher uptake (p < 0.05) in the liver (5 h = 8.96% ID g(-1); 24 h = 8.27% ID g(-1)) than IR-injected mice (5 h = 0.09% ID g(-1); 24 h = 0.08% ID g(-1)). IR-injected mice show localized uptake in the large intestine (5 h = 10.37% ID g(-1); 24 h = 0.42% ID g(-1)) with minimal uptake in other organs. Raman imaging of excised tissues correlate well with biodistribution data. These results suggest that the topical application of SERS nanoparticles in the mouse colon appears to minimize their systemic distribution, thus avoiding potential toxicity and supporting the clinical translation of Raman spectroscopy as an endoscopic imaging tool.

Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation and downregulated in the aged brain. In addition to revitalizing other aged tissues, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

Microglial Malfunction: The Third Rail in the Development of Alzheimer's Disease

Studies of Alzheimers disease (AD) have predominantly focused on two major pathologies: amyloid-β (Aβ) and hyperphosphorylated tau. These misfolded proteins can accumulate asymptomatically in distinct regions over decades. However, significant Aβ accumulation can be seen in individuals who do not develop dementia, and tau pathology limited to the transentorhinal cortex, which can appear early in adulthood, is usually clinically silent. Thus, an interaction between these pathologies appears to be necessary to initiate and propel disease forward to widespread circuits. Recent multidisciplinary findings strongly suggest that the third factor required for disease progression is an aberrant microglial immune response. This response may initially be beneficial; however, a maladaptive microglial response eventually develops, fueling a feed-forward spread of tau and Aβ pathology.