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A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKaand high affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rsin vitro. Nicotine, with lower pKaand α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligandsin vivoin male and female mouse brain and identifying the trapping brain organellein vitroas Golgi satellites (GSats). Two PET18F-labeled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKaand affinity and [18F]Nifene with nicotine-like pKaand affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4β2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to α4β2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in β2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on β2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributionsin vivoconsistent with only [18F]2-FA trapping in GSats. These results are further supported byin vitrofindings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combiningin vitroandin vivoimaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4β2R-containing GSats, which results in very slow release long after nicotine is gone after smoking.SIGNIFICANCE STATEMENTMechanisms of nicotine addiction remain poorly understood. An earlier study usingin vitromethods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in α4β2R-containing acidic vesicles. Using a fluorescent-labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were α4β2R-containing Golgi satellites (GSats).In vivoPET imaging with F-18-labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combiningin vitroandin vivoimaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.

Development of positron emission tomography (PET) radiotracers that bind with high-affinity to α4β2-type nicotinic receptors (α4β2Rs) allows for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. One challenge associated with preclinical PET imaging involves the lack of true tissue reference regions free of specific tracer binding in the rodent brain, impeding accurate quantification of the tracer binding potential. Here, we investigate the use of an image-derived arterial input function for kinetic analysis of radiotracer binding in male and female mice. Two radiotracers were explored in this study: 2-[18F]FA85380 (2-FA), which displays similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, which displays similar pKa and binding affinity to nicotine. For both radiotracers, time-activity curves of the left ventricle of the heart displayed similar standardized uptake values (SUVs) across wild type mice, mice lacking the β2 subunit for tracer binding, and acute nicotine-treated mice, whereas typical reference tissue SUVs displayed high variation between groups. Binding potential values estimated from a two-tissue compartment model (2TCM) fit of the data with the image-derived input function were significantly higher than estimates from reference tissue-based estimations. Rate constants of radiotracer dissociation were very slow for 2-FA and very fast for Nifene, similar to the in vitro dissociation rates reported for varenicline and nicotine, respectively. We conclude that use of an image-derived input function for kinetic modeling of nicotinic PET ligands improves quantification compared to reference tissue-based methods, and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.

Herein, we report the development of an 18 F-labeled, activity-based small-molecule probe targeting the cancer-associated serine hydrolase NCEH1. We undertook a focused medicinal chemistry campaign to simultaneously preserve potent and specific NCEH1 labeling in live cells and animals, while permitting facile 18 F radionuclide incorporation required for PET imaging. The resulting molecule, [18 F]JW199, labels active NCEH1 in live cells at nanomolar concentrations and greater than 1000-fold selectivity relative to other serine hydrolases. [18 F]JW199 displays rapid, NCEH1-dependent accumulation in mouse tissues. Finally, we demonstrate that [18 F]JW199 labels aggressive cancer tumor cells in vivo, which uncovered localized NCEH1 activity at the leading edge of triple-negative breast cancer tumors, suggesting roles for NCEH1 in tumor aggressiveness and metastasis.

To identify the optimal threshold insup18/supF-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pOsub2/subEPRI) as ground truth for hypoxia, defined by pOsub2/sub[Formula: see text] 10 mmHg.Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2 h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of Ksuptrans/supand vsube/subwere generated to model tumor vascular properties. Images from PET/EPR/MRI were co-registered and resampled to isotropic 0.5 mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pOsub2/subEPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient ([Formula: see text]) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pOsub2/subEPRI.FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 [Formula: see text] 0.2) were SUV [Formula: see text] 1.4 [Formula: see text] SUVsubmean/suband SUV [Formula: see text] 0.6 [Formula: see text] SUVsubmax/sub. Weak-to-moderate correlations (|[Formula: see text]|lt; 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (Ksuptrans/sup) or fractional extracellular-extravascular space (vsube/sub) from DCE-MRI.This is the first in vivo comparison of FMISO uptake with pOsub2/subEPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control.

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membranepermeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKa and high-affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rs in vitro. Nicotine, with lower pKa and α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18F-labelled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKa and affinity and [18F]Nifene with nicotine-like pKa and affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4β2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to α4β2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in β2 subunit knockout mice or by acute nicotine injections demonstrating binding to sites on β2-containing receptors. Chloroquine, which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributions in vivo consistent with only [18F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4β2R-containing GSats, which results in very slow release long after nicotine is gone after smoking.SignificanceMechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in α4β2R-containing acidic vesicles. Using a fluorescent labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were α4β2R-containing Golgi satellites. In vivo PET imaging with F-18 labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.

PURPOSE: To enhance the spatial accuracy of fluorine 18 ((18)F) misonidazole (MISO) PET imaging of hypoxia by using dynamic contrast-enhanced (DCE) MR images as a basis for modifying PET images and by using electron paramagnetic resonance (EPR) partial oxygen pressure (pO(2)) as the reference standard. MATERIALS AND METHODS: Mice (n = 10) with leg-borne MCa4 mammary carcinomas underwent EPR imaging, T2-weighted and DCE MRI, and (18)F-MISO PET/CT. Images were registered to the same space for analysis. The thresholds of hypoxia for PET and EPR images were tumor-to-muscle ratios greater than or equal to 2.2 mm Hg and less than or equal to 14 mm Hg, respectively. The Dice similarity coefficient (DSC) and Hausdorff distance (d(H)) were used to quantify the three-dimensional overlap of hypoxia between pO(2) EPR and (18)F-MISO PET images. A training subset (n = 6) was used to calculate optimal DCE MRI weighting coefficients to relate EPR to the PET signal; the group average weights were then applied to all tumors (from six training mice and four test mice). The DSC and d(H) were calculated before and after DCE MRI–corrected PET images were obtained to quantify the improvement in overlap with EPR pO(2) images for measuring tumor hypoxia. RESULTS: The means and standard deviations of the DSC and d(H) between hypoxic regions in original PET and EPR images were 0.35 mm ± 0.23 and 5.70 mm ± 1.7, respectively, for images of all 10 mice. After implementing a preliminary DCE MRI correction to PET data, the DSC increased to 0.86 mm ± 0.18 and the d(H) decreased to 2.29 mm ± 0.70, showing significant improvement (P < .001) for images of all 10 mice. Specifically, for images of the four independent test mice, the DSC improved with correction from 0.19 ± 0.28 to 0.80 ± 0.29 (P = .02), and the d(H) improved from 6.40 mm ± 2.5 to 1.95 mm ± 0.63 (P = .01). CONCLUSION: Using EPR information as a reference standard, DCE MRI information can be used to correct (18)F-MISO PET information to more accurately reflect areas of hypoxia. Keywords: Animal Studies, Molecular Imaging, Molecular Imaging-Cancer, PET/CT, MR-Dynamic Contrast Enhanced, MR-Imaging, PET/MR, Breast, Oncology, Tumor Mircoenvironment, Electron Paramagnetic Resonance Supplemental material is available for this article. © RSNA, 2021

This study serves as a proof of concept for multimodal oxygen imaging in developing an algorithm to make fluorine 18 misonidazole PET more comparable with quantitative electron paramagnetic resonan...

The measurement of extracellular pH (pHe) has significant clinical value for pathological diagnoses and for monitoring the effects of pH-altering therapies. One of the major problems of measuring pHe with a relaxation-based MRI contrast agent is that the longitudinal relaxivity depends on both pH and the concentration of the agent, requiring the use of a second pH-unresponsive agent to measure the concentration. Here we tested the feasibility of measuring pH with a relaxation-based dendritic MRI contrast agent in a concentration-independent manner at clinically relevant field strengths. The transverse and longitudinal relaxation times in solutions of the contrast agent (GdDOTA-4AmP)44-G5, a G5–PAMAM dendrimer-based MRI contrast agent in water, were measured at 3 T and 7 T magnetic field strengths as a function of pH. At 3 T, longitudinal relaxivity (r1) increased from 7.91 to 9.65 mM−1 s−1 (on a per Gd3+ basis) on changing pH from 8.84 to 6.35. At 7 T, r1 relaxivity showed pH response, albeit at lower mean values; transverse relaxivity (r2) remained independent of pH and magnetic field strengths. The longitudinal relaxivity of (GdDOTA-4AmP)44-G5 exhibited a strong and reversible pH dependence. The ratio of relaxation rates R2/R1 also showed a linear relationship in a pH-responsive manner, and this pH response was independent of the absolute concentration of (GdDOTA-4AmP)44-G5 agent. Importantly, the nanoprobe (GdDOTA-4AmP)44-G5 shows pH response in the range commonly found in the microenvironment of solid tumors. Copyright © 2015 John Wiley & Sons, Ltd.

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

In a low-resource setting, an ideal disease diagnostic modality is an eye visualization assay that can be performed in minutes by untrained personnel. This paper reports the development of a self-catalytic colorimetric assay to detect the existence of cervical cancer cells by naked eye. The assay consists of a target specific initiator/catalyst, an acid self-catalytic system, and a pH indicator solution. By taking the advantage that trace amount of nucleus staining molecule, hemalum, can function as the initiator, a stained cervical cancer cell with an enlarged nucleus can initiate the self-catalytic reaction in a defined reaction condition to produce a significant amount of acid, which leads to a color change of a pH indicator solution, thereby indicating the presence of the cancer cell. Amplification of at least a trillion fold was achieved within 1.5 min. Due to the minimum engagement of instruments, simplicity and low cost, the assay is a potential tool in clinical to function as a “yes/no” check to facilitate quick decisions for cervical cancer screening. By alternating the method whereby the initiator is associated with the target, the assay can be expanded to screen and detect protein or nucleic acid based biomarkers related to other epidemic diseases in the resource-limited settings.

Aim: The authors have investigated the usefulness of in vivo chemical exchange saturation transfer MRI for detecting gliomas using a dual-modality imaging contrast agent. Materials & methods: A paramagnetic chemical exchange saturation transfer MRI contrast agent, Eu-1,4,7,10-tetraazacclododecane-1,4,7,10-tetraacetic acid-Gly4 and a fluorescent agent, DyLight® 680, were conjugated to a generation 5 polyamidoamine dendrimer to create the dual-modality, nano-sized imaging contrast agent. Results: The agent was detected with in vivo chemical exchange saturation transfer MRI in an U87 glioma model. These results were validated using in vivo and ex vivo fluorescence imaging. Conclusion: This study demonstrated the merits of using a nano-sized imaging contrast agent for detecting gliomas and using a dual-modality agent for detecting gliomas at different spatial scales. Original submitted 9 January 2012; Revised submitted 18 April 2012; Published online 14 August 2012

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that effect gene regulation. To know the interaction of aliphatic cap groups with HDACs, cyclic tetrapeptide and bicyclic peptide disulfide hybrids were synthesized without aromatic ring in their macrocyclic framework. Benzene ring of l -Phe in chlamydocin was replaced with several aliphatic amino acids and also a fused bicyclic tetrapeptide was synthesized by ring closing metathesis using Grubb’s first generation catalyst. The inhibitory activities of the cyclic peptides against histone deacetylase enzymes were evaluated, which demonstrated most of them are interesting candidates as anticancer agents.

A water-soluble l-lysine dendrimer (6) with links to porphyrins and a fullerene was synthesized, in which the dendrimer surface was modified with carboxylate groups. The aqueous solution of 6 showed the Soret band characteristic of the aggregated porphyrins in the UV−vis spectra; however, this aggregate was dissociated by the addition of 2-hydroxypropyl-β-cyclodextrin (10 wt % in H2O). TEM and AFM showed the formation of submicron-scale particles of 6. TEM showed the spherical particles of 6 with almost uniform size (100−110 nm) in the presence of the cyclodextrin. AFM showed the oval particles of 6 in the absence of the cyclodextrin. DLS measurement indicated that cyclodextrin dissociated the aggregated structure of 6 in H2O. Thus, a large number of porphyrins and fullerenes were successfully accumulated with a narrow size distribution via the particle formation of the amphiphilic dendrimer.

[60]-Fullerene functionalized amino acids with 4–6 methylene spacers from α-carbon to the nitrogen atom of fulleropyrolidine and corresponding multifullerene peptides have been synthesized.

Using copper (II) ion as a model pollutant, we report a new bioremediation concept, which involves the use of green algae Chlamydomonas reinhardtii to efficiently collect copper ions from the solution, followed by encapsulating copper loaded algae with silica, thus reducing the bioavailability of copper ions in the solution. Specifically, the potential of Chlamydomonas reinhardtii as an active copper (II) absorbent was demonstrated by quantifying and characterizing the copper uptake rate, capacity, efficacy, as well as copper retention from C. reinhardtii. Subsequently, a method of encrusting copper loaded C. reinhardtii with silica was developed, taking the advantage that the presences of high abundant polysaccharides and glycoproteins on the cell walls, as well as the presence of (3-amino-propyl) trimethoxysilane (APS) can function as nucleation center for silicification process of tetramethyl orthosilicate (TMOS). Both fluorescence imaging and scanning electron microscope (SEM) imaging confirmed the silica encrustation. It is expected that silica encrustation of algae has the potential for in situ remediation of various contaminants in a wide range of environments while providing long-term stabilization and diminishing the bioavailability of the contaminants.

Background Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities. Methods and Results Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors. Conclusion EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes.

The naturally occurring cyclic tetrapeptide, chlamydocin, originally isolated from fungus Diheterospora chlamydosphoria, consists of α-aminoisobutyric acid, l-phenylalanine, d-proline and an unusual amino acid (S)-2-amino-8-((S)-oxiran-2-yl)-8-oxooctanoic acid (Aoe) and inhibits the histone deacetylases (HDACs), a class of regulatory enzymes. The epoxyketone moiety of Aoe is the key functional group for inhibition. The cyclic tetrapeptide scaffold is supposed to play important role for effective binding to the surface of enzymes. In place of the epoxyketone group, hydroxamic acid and sulfhydryl group have been applied to design inhibitor ligands to zinc atom in catalytic site of HDACs. In the research for more potent HDAC inhibitors, we replaced the epoxyketone moiety of Aoe with different functional groups and synthesized a series of chlamydocin analogs as HDAC inhibitors. Among the functional groups, methoxymethylketone moiety showed as potent inhibition as the hydroxamic acid. On the contrary, we confirmed that borate, trifruoromethylketone, and 2-aminoanilide are almost inactive in HDAC inhibition.

Introduction Enantiomerically pure aliphatic amino acids having easily modifiable functional groups at the end of the side chain on α-carbon atom are highly interesting synthetic targets as the terminal functional groups can be changed to the required groups which provide a series of non-poteinogenic amino acids [1]. As the double bond is a masked functional group and is stable to most acidic and basic reaction conditions, the classical reactivity of alkenes towards addition, oxidation, etc., can be used for the side chain modification. So it is worthy to synthesize α-amino acids containing side chains with a terminal double bond, 2-amino-(n-1)-alkenoic acids (Aens). As a precursor, it can be easily converted to various amino acids having different side chain functionality. In fact several methods are available for the synthesis of specific ω-unsaturated α-amino acids [2,3]. Here we report a convenient method, for the synthesis of Boc-2-amino-(n-1)-alkenoic acids (Boc-Aen-OH, n = 5-9) from commercially available reagents, diethyl Boc-aminomalonate and ω-bromoalkenes. We also explored the possibility of their use in the synthesis of some new zinc ligands for chlamydocin type cyclic tetrapeptide scaffold in order to develop histone deacetylase inhibitors as anti-cancer agents with optimized pharmacokinetic properties.

A series of chlamydocin analogs with various carbonyl functionalities were designed and synthesized as histone deacetylase (HDAC) inhibitors. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone surrogate in the side chain which makes it irreversible inhibitor of HDACs, whereas apicidins are a class of cyclic tetrapeptides that contain an ethylketone moiety as zinc ligand. We replaced the epoxyketone moiety of chlamydocin with several ketones and aldehyde to synthesize potent reversible and selective HDAC inhibitors. The inhibitory activity of the cyclic tetrapeptides against histone deacetylase enzymes were evaluated and the result showed most of them are potent inhibitors. Some of them have remarkable selectivity among the HDACs.