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This work focuses on the development of specific substrates for estrogen sulfotransferase (SULTIE1) to produce molecular imaging probes for this enzyme. SULTIE1 is a key enzyme in estrogen homeostasis, playing a central role in the prevention and development of human disease. In vitro sulfation assays showed alkyl and aryl substitutions to a fused heterocyclic system modeled after [beta]-naphthol ([beta]N), based on compounds that interact with the estrogen receptor, rendered several molecules with enhanced specificity for SULTIE1 over [SULT1A1.sup.*]1, [SULT1A1.sup.*]2, SULT1A3, and SULT2A1. Several 6-hydroxy-2-arylbenzothiazoles tested demonstrated excellent affinity--[V.sub.max]/[K.sub.m] ratios--and specificity for SULT1E1. [K.sub.m] values ranged from 0.12-2.36 [micro]M. A strong correlation was observed between polarity of the 4'-sustituent on the 2-aryl moiety (Hammett [[sigma].sub.p]) and the Iog([V.sub.max]/[K.sub.m]) (r = 0.964). Substrate sensitivity is influenced by the acidity of the 6-phenolic group demonstrated by correlating its [sup.1]H NMR chemical shift ([delta]OH) with the Iog([V.sub.max]/[K.sub.m]) (r = 0.963). Acidity is mediated by the electron withdrawing capacity of the 4'-substituent outlined by the correlation of the C-2 [sup.13]C NMR chemical shift ([[delta].sub.C2]) with the Iog([V.sub.max]/[K.sub.m]) (r = 0.987). 2-[4-(Methylamino)phenyl]-6-hydroxybenzothiazole (2b) was radiolabeled with carbon-11 ([sup.11]C-(2b)) and used in vivo for microPET scanning and tissue metabolite identification. High PET signal was paralleled with the presence of radiolabeled [sup.11]C-(2b)-6-O-sulfate and the SULTIE1 protein detected by western blot. Because this and other members of this family presenting specificity for SULT1E1 can be labeled with carbon-11 or fluorine-18, in vivo assays of SULT1E1 functional activity are now feasible in humans. molecular imaging probes | positron emission tomography | Pittsburgh Compound B | PIB | [sup.18]F-Flutemetamol doi/10.1073/pnas.0914904107

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway that recycles products of DNA degradation, dCK phosphorylates and therefore activates nucleoside analog prodrugs frequently used in cancer, autoimmunity, and viral infections. In contrast to its well established therapeutic relevance, the biological function of dCK remains enigmatic. Highest levels of dCK expression are found in thymus and bone marrow, indicating a possible role in lymphopoiesis. To test this hypothesis we generated and analyzed dCK knockout (KO) mice. dCK inactivation selectively and profoundly affected T and B cell development. A 90-fold decrease in thymic cellularity was observed in the dCK KO mice relative to wild-type littermates. Lymphocyte numbers in the dCK KO mice were 5- to 13-fold below normal values. The severe impact of dCK inactivation on lymphopoiesis was unexpected given that nucleoside salvage has been thought to play a limited, 'fine-tuning' role in regulating deoxyribonucleotide triphosphate pools produced by the de novo pathway. The dCK KO phenotype challenges this view and indicates that, in contrast to the great majority of other somatic cells, normal lymphocyte development critically requires the deoxyribonucleoside salvage pathway. combined immunodeficiency | deoxyribonucleoside salvage | dNTP metabolism | immune development | T and B lymphocytes www.pnas.org/cgi/doi/10.1073/pnas.0913900107

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and [sup.18]F-labeled 1-(2'-deoxy-2'-fluoroarabinofuranosyl) cytosine] ([[sup.18]F]FAC), a PET probe recently developed by our group. We show that [[sup.18]F]FAC is a DCK substrate with an affinity similar to that of dFdC. In vitro, accumulation of [[sup.18]F]FAC in murine and human leukemia cell lines is critically dependent on DCK activity and correlates with dFdC sensitivity. In mice, [[sup.18]F]FAC accumulates selectively in DCK-positive vs. DCK-negative tumors, and [[sup.18]F]FAC microPET scans can predict responses to dFdC. We suggest that [[sup.18]F]FAC PET might be useful for guiding treatment decisions in certain cancers by enabling individualized chemotherapy. [[sup.18]F]FAC | individualized chemotherapy | molecular imaging | deoxycytidine kinase

The biodistribution profiles in mice of two pyrrole-imidazole polyamides were determined by PET. Pyrrole-imidazole polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor-DNA interfaces, and modulate gene expression pathways in cell culture experiments. The [sup.18]F-radiolabeled polyamides were prepared by oxime ligation between 4-[[sup.18]F]-fluorobenzaldehyde and a hydroxylamine moiety at the polyamide C terminus. Small animal PET imaging of radiolabeled polyamides administered to mice revealed distinct differences in the biodistribution of a 5-ring [beta]-linked polyamide versus an 8-ring hairpin, which exhibited better overall bioavailability. In vivo imaging of pyrrole-imidazole polyamides by PET is a minimum first step toward the translation of polyamide-based gene regulation from cell culture to small animal studies. biodistribution | fluorine-18 | oxime | radiosynthesis

4-[F-18]fluoro-N-{2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl}-N-(2-pyridinyl)benzamide, a selective serotonin 1A (5-H[T.sub.1A]) molecular imaging probe, was used in conjunction with positron emission tomography (PET) for quantification of 5-H[T.sub.1A] receptor densities in the living brains of Alzheimer's disease patients (ADs) (n = 8), subjects with mild cognitive impairment (n = 6), and controls (n = 5). ADs had receptor densities significantly decreased in both hippocampi (binding potential: controls 1.62 [+ or -] 0.07; ADs 1.18 [+ or -] 0.26) and also in raphe nuclei (controls 0.63 [+ or -] 0.09; ADs 0.37 [+ or -] 0.20). When volume losses are included, 5-H[T.sub.1A] losses are even more severe (i.e., average mean decreases of 24% in mild cognitive impairment patients and 49% in ADs). A strong correlation of 5-H[T.sub.1A] receptor decreases in hippocampus with worsening of clinical symptoms (Mini Mental State Exam scores) was also found. Moreover, these decreases in 5-H[T.sub.1A] receptor measures correlate with decreased glucose utilization as measured with 2-deoxy-2-[F-18] fluoro-D-glucose PET in the brains of ADs (standardized uptake values; globally: controls 0.89 [+ or -] 0.04, ADs 0.72 [+ or -] 0.04; posterior cingulate gyrus: controls 1.05 [+ or -] 0.09, ADs 0.79 [+ or -] 0.11). They also inversely correlate with increased neuropathological loads measured with 2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene) malononitrile PET in several neocortical regions in the same subjects. The in vivo observations were confirmed independently by in vitro digital autoradiography with 4-[F-18]fluoro-N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) benzamide and 2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl} ethylidene)malononitrile on brain tissue specimens from two ADs and three nondemented subjects. [F-18]MPPF | brain 5-H[T.sub.1A] receptors | neuronal loss | positron emission tomography

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the HSV1-tk reporter gene in living mice. There is a significant positive correlation between the percent injected dose of FGCV retained per gram of liver and the levels of hepatic HSV1-tk reporter gene expression ([r.sup.2] [greater than] 0.80). Over a similar range of HSV1-tk expression in vivo, the percent injected dose retained per gram of liver was 0-23% for ganciclovir and 0-3% for FGCV. Repeated, noninvasive, and quantitative imaging of PET reporter gene expression should be a valuable tool for studies of human gene therapy, of organ/cell transplantation, and of both environmental and behavioral modulation of gene expression in transgenic mice.

Glucose is a major metabolic substrate required for cancer cell survival and growth. It is mainly imported into cells by facilitated glucose transporters (GLUTs). Here we demonstrate the importance of another glucose import system, the sodium-dependent glucose transporters (SGLTs), in pancreatic and prostate adenocarcinomas, and investigate their role in cancer cell survival. Three experimental approaches were used: (i) immunohistochemical mapping of SGLT1 and SGLT2 distribution in tumors; (ii) measurement of glucose uptake in fresh isolated tumors using an SGLT-specific radioactive glucose analog, α-methyl- 4-deoxy-4-[18F]fluoro-D-glucopyranoside (Me4FDG), which is not transported by GLUTs; and (iii) measurement of in vivo SGLT activity in mouse models of pancreatic and prostate cancer using Me4FDG-PET imaging. We found that SGLT2 is functionally expressed in pancreatic and prostate adenocarcinomas, and provide evidence that SGLT2 inhibitors block glucose uptake and reduce tumor growth and survival in a xenograft model of pancreatic cancer. We suggest that Me4FDG-PET imaging may be used to diagnose and stage pancreatic and prostate cancers, and that SGLT2 inhibitors, currently in use for treating diabetes, may be useful for cancer therapy. [ABSTRACT FROM AUTHOR]

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluo-roethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-ß (Aß) and other amyloid aggregates present in Alzheimer's disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aß aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aß aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aß peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. [ABSTRACT FROM AUTHOR]

We have developed an all-electronic digital microfluidic device for microscale chemical synthesis in organic solvents, operated by electrowetting-on-dielectric (EWOD). As an example of the principles, we demonstrate the multistep synthesis of [(18)F]FDG, the most common radiotracer for positron emission tomography (PET), with high and reliable radio-fluorination efficiency of [(18)F]FTAG (88 ± 7%, n = 11) and quantitative hydrolysis to [(18)F]FDG (> 95%, n = 11). We furthermore show that batches of purified [(18)F]FDG can successfully be used for PET imaging in mice and that they pass typical quality control requirements for human use (including radiochemical purity, residual solvents, Kryptofix, chemical purity, and pH). We report statistical repeatability of the radiosynthesis rather than best-case results, demonstrating the robustness of the EWOD microfluidic platform. Exhibiting high compatibility with organic solvents and the ability to carry out sophisticated actuation and sensing of reaction droplets, EWOD is a unique platform for performing diverse microscale chemical syntheses in small volumes, including multistep processes with intermediate solvent-exchange steps.